Germinal center kinase proteins, compositions and methods of use

ABSTRACT

The present invention provides compositions and methods for modulating cell proliferation, survival, morphology, and migration. Nucleic acids encoding proteins and proteins so encoded which are capable of modulating proliferation, survival, morphology and migration in mammalian cells are provided. Compositions and methods for the treatment of disorders related to cell proliferation, survival, morphology and migration are also provided. Prophylactics and methods for the prevention of such disorders are also provided. Also provided are compositions and methods for diagnostic and prognostic determination of such disorders. Further provided are assays for the identification of bioactive agents capable of modulating proliferation, survival, morphology and migration in mammalian cells.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingcell proliferation, survival, morphology, and migration. Nucleic acidsencoding proteins and proteins so encoded which are capable ofmodulating proliferation, survival, morphology and migration inmammalian cells are provided. Compositions and methods for the treatmentof disorders related to cell proliferation, survival, morphology andmigration are also provided. Prophylactics and methods for theprevention of such disorders are also provided. Also provided arecompositions and methods for diagnostic and prognostic determination ofsuch disorders. Further provided are assays for the identification ofbioactive agents capable of modulating proliferation, survival,morphology and migration in mammalian cells.

BACKGROUND OF THE INVENTION

Cells cycle through various stages of growth, starting with the M phase,where mitosis and cytoplasmic division (cytokinesis) occurs. The M phaseis followed by the G1 phase, in which the cells resume a high rate ofbiosynthesis and growth. The S phase begins with DNA synthesis, and endswhen the DNA content of the nucleus has doubled. The cell then enters G2phase, which ends when mitosis starts, signaled by the appearance ofcondensed chromosomes. Terminally differentiated cells are arrested inthe G1 phase, and no longer undergo cell division.

The hallmark of a malignant cell is uncontrolled proliferation. Thisphenotype is acquired through the accumulation of gene mutations, themajority of which promote passage through the cell cycle. Cancer cellsignore growth regulatory signals and remain committed to cell division.Classic oncogenes, such as ras, lead to inappropriate transition from G1to S phase of the cell cycle, mimicking proliferative extracellularsignals. Cell cycle checkpoint controls ensure faithful replication andsegregation of the genome. The loss of cell cycle checkpoint controlresults in genomic instability, greatly accelerating the accumulation ofmutations which drive malignant transformation. Thus, modulating cellcycle checkpoint pathways and other such pathways with therapeuticagents could exploit the differences between normal and tumor cells,both improving the selectivity of radio- and chemotherapy, and leadingto novel cancer treatments. As another example, it would be useful tocontrol entry into apoptosis.

On the other hand, it is also sometimes desirable to enhanceproliferation of cells in a controlled manner. For example,proliferation of cells is useful in wound healing and where growth oftissue is desirable. Thus, identifying modulators which promote, enhanceor deter the inhibition of proliferation is desirable.

Proteins of general interest that have been reported on include kinases.The Ste20 family of kinases can be divided into two structurallydistinct subfamilies. The first subfamily contains a C-terminalcatalytic domain and an N-terminal binding site for the small G proteinsRac1 and Cdc42 (Herskowitz, Cell, 80:187-197 (1995)). The yeastserine/threonine kinase Ste20 and its mammalian homologue, p21 ActivatedKinase 1 (PAK1), belong to this subfamily. Ste20 initiates amitogen-activated protein kinase (MAPK) cascade that includes Ste11(MAPKKK), Ste7 (MAPKK), and FUS3/KSS1 (MAPK) in response to activationof the small G protein Cdc42, as well as signals from thehetero-trimeric G proteins coupled to pheromone receptors (Herskowitz,Cell, 80:187-197 (1995)). Similar to Ste20 , PAK1 has been reported tobe a Cdc42 and Rac1 effector molecule and specifically regulates thec-Jun N-terminal kinase (JNK) pathway, one of the mammalian MAPKpathways (Bagrodia, et. al., J. Biol. Chem., 270:27995-27998 (1995);Kyriakis, et al., J. Biol. Chem., 271:24313-24316 (1996)). The JNKpathway is activated by a variety of stress inducing agents, includingosmotic and heat shock, UV irradiation, protein inhibitors andpro-inflammatory cytokines such as tumor necrosis factor (TNF) (Ip, etal., Curr. Opin. Cell Biol., 10:205-219 (1998)). JNKs are activatedthrough threonine and tyrosine phosphorylation by MEK4 and MEK7 (MAPKK),which are in turn phosphorylated and activated by MAPKKKs including MEKkinase 1 (MEKK1), and mixed lineage kinases MLK2 and MLK3 (Ip, et al.,Curr. Opin. Cell Biol., 10:205-219 (1998)). In addition to theactivation of the JNK pathway, PAK1 has also been reported to be aregulator of the actin cytoskeleton (Sells, et al., Curr. Biol.,7:202-210 (1997)).

The second subgroup of Ste20 family of kinases is represented by thefamily of germinal center kinases (GCK) (Kyriakis, J. Biol. Chem.,274:5259-5262 (1999)). In contrast to Ste20 and PAK1, GCK family membershave an N-terminal kinase domain and a C-terminal regulatory region.Many GCK family members, including GCK, germinal center kinase relatedprotein (GCKR), meatopoietic protein kinase (HPK) 1, GCK-like kinase(GLK), HPK/GCK-like kinase (HGK) and NCK interacting kinase (NIK), havealso been reported to activate the JNK pathway when overexpressed in 293cells (Pombo, et al., Nature, 377:750-754 (1995); Shi, et al., J. Biol.Chem., 272:32102-32107 (1997); Kiefer, et al., EMBO J. 15:7013-7025(1996); Diener, et al., Proc. Natl. Acad. Sci. USA, 94:9687-9692 (1997);Yao, et al., J. Biol. Chem., 274:2118-2125 (1999); Su, et al., EMBO J.,16:1279-1290 (1997)). Among those, GCK and GCKR have been implicated inmediating TNF-induced JNK activation through TNF receptor associatedfactor 2 (Traf2) (Pombo, et al., Nature, 377:750-754 (1995); Diener, etal., Proc. Natl. Acad. Sci. USA, 94:9687-9692 (1997); Yuasa, et al., J.Biol. Chem., 273:22681-22692 (1998)). NCK interacting kinase (NIK)interacts with the SH2-SH3 domain containing adapter protein NCK and hasbeen proposed to link protein tyrosine kinase signals to JNK activation(Su, et al., EMBO J., 16:1279-1290 (1997)).

A kinase related to TNIK has been reported on. MINK(misshapen/NIKs-related kinase) protein and nucleic acid have beenpreviously described (Ippeita et. al., FEBS Letters, 469:19-23, 2000).MINK1 is a gck kinase family member which is upregulated during braindevelopment (Ippeita et. al., FEBS Letters, 469:19-23, 2000).

One study reports on a GCK family kinase from Dictyostelium that canphosphorylate Severin in vitro. (Eichinger, et al., J. Biol. Chem.,273:12952-12959 (1998)). Severin is an F-actin fragmenting and cappingenzyme that regulates Dictyostelium motility. TNIK, a mammalian GCK, hasbeen shown to regulate the cytoskeleton, particularly to destabilizeF-actin (Fu et al., JBC 274:30729-30737, 1999).

The Rho, rac and cdc42 small GTPases have been shown to regulate actinpolymerization and the formation of multimolecular focal complexes (forexample, see Nobes et al., Cell 81:53-62, 1995, and references therein;incorporated herein by reference). Further, PAK1 has been shown toregulate actin cytoskeleton organization, possibly through thephosphorylation and inhibition of the myosin light chain kinase Sanderset al., Science 283:2083-2085, 1999).

In addition, intracellular signaling mechanisms affecting cytoskeletalorganization and underlying cell migration in response to extracellularcues have been studied in some detail (for review, see Maghazachi etal., Int. J. Biochem. Cell Biol. 32:931-943, 2000).

Several kinases and other intracellular signaling molecules have alsobeen implicated in the control of apoptosis and cell survival inmammalian cells. For example, the JNK family of kinases has beenimplicated in both apoptosis and cell survival, the particular effectbeing dependent on the cellular context (for review, see Ip et al.,Curr. Opin. Cell Biol. 10:205-219, 1998).

The role of GCKs in the immune system is of particular interest.Although GCKs are expressed widely, in B lymphocytic follicular tissue,GCK expression is largely restricted to the germinal center (Katz etal., JBC 269:16802-16809, 1994). In germinal centers, B lymphocytesundergo differentiation and selection, which is induced in part byligands including members of the TNF family. These ligands activate GCKswhich in turn activate other protein kinases that induce lymphocytedevelopment (reviewed in Kyriakis, JBC 274:5259-5262, 1999).

The integrity of intracellular signal transduction pathways and theirappropriate regulation is essential for B cell and T cell developmentand function. An understanding of these signaling pathways is thereforedesirable to provide means for therapeutically modulating lymphocytefunction in a variety of disorders characterized by hyper immuneresponses (e.g. auto-immune disorders) or hypo immune responses (e.g.immunodeficiency disorders). Such understanding is also desirable toprovide for the modulation of normal but undesirable immune responses,for example following transplant immunosuppressive agents are desirable.

The modulation of signal transduction, proliferation, apoptosis,morphological change, and migration in mammalian cells is desirable, forexample for the treatment of cancer, immune dysfunction, and forimmunosuppression. Accordingly, compositions and methods for modulatingthese processes in mammalian cells are desirable.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for modulatingproliferation, survival, migration, morphology, cytoskeletalorganization and intracellular signal transduction in mammalian cells.Nucleic acids encoding proteins and proteins so encoded which arecapable of modulating proliferation, survival, migration, morphology,cytoskeletal organization and intracellular signal transduction inmammalian cells are provided. Compositions and methods for the treatmentof disorders related to cell proliferation, survival, morphology andmigration are also provided. Prophylactics and methods for theprevention of such disorders are also provided. Also provided arecompositions and methods for diagnostic and prognostic determination ofsuch disorders. Further provided are assays for the identification ofbioactive agents capable of modulating signal transduction,proliferation, survival, morphology and migration in mammalian cells.

Accordingly, the present invention provides MINK3 nucleic acids,including nucleic acids encoding MINK3 protein, which are capable ofmodulating proliferation, survival, migration, morphology, cytoskeletalorganization and intracellular signal transduction in mammalian cells.Also provided herein are MINK3 antisense nucleic acids which are capableof modulating proliferation, survival, migration, morphology,cytoskeletal organization and intracellular signal transduction inmammalian cells. Also provided herein are MINK3 proteins, includingdominant negative MINK3 proteins, which are capable of modulatingproliferation, survival, migration, morphology, cytoskeletalorganization and intracellular signal transduction in mammalian cells.

MINK (misshapen/NIKs-related kinase) proteins and nucleic acids havinghomology to the MINK3 proteins and nucleic acids described herein havebeen previously described (Ippeita et. al., FEBS Letters, 469:19-23,2000). For example, MINK1 is a gck kinase family member which isupregulated during brain development (Ippeita et. al., FEBS Letters,469:19-23, 2000).

In one aspect, the invention is directed to MINK3 proteins. In anotheraspect, the invention is directed to recombinant MINK3 nucleic acids,including nucleic acids encoding MINK3 proteins. In a further aspect,the invention is directed to recombinant MINK3 antisense nucleic acidscomprising nucleic acid sequences complementary to the nucleic acidsequences of MINK3 nucleic acids or fragments thereof.

In a preferred embodiment of the invention, the MINK3 nucleic acidcomprises a nucleic acid sequence selected from the group consisting ofthe nucleic acid sequences set forth in SEQ ID NOs: 1, 3, and 5, orcomplements thereof.

In another preferred embodiment, the MINK3 nucleic acid comprises anucleic acid sequence having at least about 90% identity, morepreferably at least about 95% identity to a nucleic acid sequenceselected from the group consisting of the nucleic acid sequences setforth in SEQ ID NOs:1, 3, and 5, or complements thereof.

In another preferred embodiment, the MINK3 nucleic acid will hybridizeunder high stringency conditions to a nucleic acid comprising a nucleicacid sequence selected from the group consisting of the nucleic acidsequences set forth in SEQ ID NOs:1, 3, and 5, or complements thereof.

In a preferred embodiment, the MINK3 antisense nucleic acid comprises anucleic acid sequence complementary to the nucleic acid sequence setforth in SEQ ID NO:1, more preferably to the nucleic acid sequence setforth by a fragment of SEQ ID NO:1, more preferably to the nucleic acidsequence set forth by nucleotides 2804-3187 in SEQ ID NO:1.

In a preferred embodiment, the MINK3 antisense nucleic acid has anucleic acid sequence that consists essentially of the complement of thenucleic acid sequence set forth by nucleotides 2804-3187 in SEQ ID NO:1.

In a preferred embodiment, the MINK3 antisense nucleic acid hybridizesto MINK3a and MINK1 nucleic acids.

In a preferred embodiment, the MINK3 nucleic acid comprises a nucleicacid sequence encoding MINK3 protein. Preferably MINK3 protein soencoded will bind Nck.

In a preferred embodiment, the MINK3 nucleic acid comprises a nucleicacid sequence encoding a MINK3 protein comprising an amino acid sequenceselected from the group consisting of the amino acid sequences set forthin SEQ ID NOs: 2, 4, and 6.

In a preferred embodiment, the MINK3 nucleic acid comprises a nucleicacid sequence encoding a MINK3 protein comprising an amino acid sequencehaving at least about 90% identity, more preferably at least about 95%identity, to an amino acid sequence selected from the group consistingof the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6.

In one aspect, the present invention provides MINK3 proteins encoded byMINK3 nucleic acids described herein.

In one aspect, the invention provides three isoforms of MINK3 proteinand nucleic acid, namely MINK3a, MINK3b and MINK3c, comprising aminoacid and nucleic acid sequences as described herein. MINK3b and MINK3cnucleic acids have a frameshift relative to MINK3a. As a consequence,MINK3b and MINK3c proteins are kinase dead proteins.

In one aspect, the invention provides MINK3 antisense nucleic acids. Ina preferred embodiment, the MINK3 antisense nucleic acid will inhibitgrowth factor-induced activation of an extracellular signal responsekinase (ERK), preferably EGF-induced ERK activation.

In a preferred embodiment, the MINK3 antisense nucleic acid will inhibitproliferation in a mammalian cell, preferably a cancer cell.

In a preferred embodiment, the MINK3 antisense nucleic acid will inhibitaberrant cell proliferation, for example as occurs in cancer. Preferablysuch aberrant cell proliferation involves aberrant ERK and/or JNKpathway activation.

In a preferred embodiment, the MINK3 antisense nucleic acid will inhibitgrowth factor-dependent proliferation in a mammalian cell. Preferablysuch growth factor-dependent proliferation is EGF-dependent.

In a preferred embodiment, the MINK3 antisense nucleic acid will inhibitphosphorylation of c-JUN N-terminal kinase (JNK) and/or ERK.

In a preferred embodiment, the MINK3 antisense nucleic acid will inhibitactivation of JNK and/or ERK.

In a preferred embodiment, the MINK3 antisense nucleic acid will inhibitthe JNK signal transduction pathway and/or the ERK signal transductionpathway in a mammalian cell. Preferably the mammalian cell is a cancercell and/or a lymphocyte.

In a preferred embodiment, the MINK3 antisense nucleic acid will inhibittaxol-induced cleavage of the retinoblastoma protein (Rb) and apoptosisin a mammalian cell. In a preferred embodiment, the MINK3 antisensenucleic acid will promote survival in a mammalian cell followingexposure to taxol.

In a preferred embodiment, the MINK3 antisense nucleic acid will inhibitthe transcription promoting activity of AP-1 in a mammalian cell. In apreferred embodiment, the MINK3 antisense nucleic acid will inhibittranscriptional activation by one or more AP-1 response elements.

In one embodiment, a MINK3 nucleic acid has an activity opposite to thatof a MINK3 antisense nucleic acid.

In another aspect of the invention, expression vectors are provided. Theexpression vectors comprise one or more MINK3 nucleic acids describedherein operably linked to regulatory sequences recognized by a host celltransformed with the nucleic acid. Further provided herein are hostcells comprising the vectors and MINK3 nucleic acids provided herein.Moreover, provided herein are processes for producing MINK3 proteincomprising culturing a host cell under conditions suitable forexpression of the MINK3 protein. In one embodiment, the process includesrecovering the MINK3 protein.

In one aspect, the invention is directed to MINK3 proteins.

In a preferred embodiment of the invention, the MINK3 protein comprisesan amino acid sequence selected from the group consisting of the aminoacid sequences set forth in SEQ ID NOs: 2, 4, and 6.

In another preferred embodiment, the MINK3 protein comprises an aminoacid sequence having at least about 90% identity, more preferably atleast about 95% identity to an amino acid sequence selected from thegroup consisting of the amino acid sequences set forth in SEQ ID NOs: 2,4, and 6.

In a preferred embodiment, the MINK3 protein will bind to NCK protein,such as Nck protein comprising the amino acid sequence set forth atGenbank accession number AAD13752.

In another preferred embodiment, the MINK3 protein will effect a changein morphology in a mammalian cell, preferably a cancer cell. In apreferred embodiment, the morphology affecting activity of MINK3 proteinis MEK-dependent.

In a preferred embodiment, the MINK3 protein will disrupt actinfilaments in a mammalian cell, preferably a cancer cell.

In a preferred embodiment, the MINK3 protein will phosphorylate JNKand/or ERK.

In a preferred embodiment, the MINK3 protein will activate JNK and/orERK.

In a preferred embodiment, the MINK3 protein will activate the JNKsignal transduction pathway and/or the ERK signal transduction pathwayin a mammalian cell.

In another preferred embodiment, the MINK3 protein will induce cellcycle progression and proliferation in a mammalian cell.

In a preferred embodiment, the MINK3 protein comprises a germinal centerkinase domain (GCK) (sometimes referred to herein, and in theliterature, as a “CNH” domain) as that set forth by amino acids 994-1292or 994-1290 in SEQ ID NO:2.

In a preferred embodiment, the MINK3 protein comprises a catalyticserine/threonine kinase domain, as that set forth by amino acids 25-289in SEQ ID NO:2.

In a preferred embodiment, the MINK3 protein comprises a catalytictyrosine kinase domain, as that set forth by amino acids 26-286 in SEQID NO:2.

In a preferred embodiment, the MINK3 protein provided herein comprisesan ATP-binding domain, such as that set forth by amino acids 32-54 inSEQ ID NO:2.

In one aspect, the invention is directed to dominant negative MINK3proteins. A dominant negative MINK3 protein will antagonize at least oneMINK3 protein activity.

In a preferred embodiment, the dominant negative MINK3 protein willinhibit the JNK signal transduction pathway and/or the ERK signaltransduction pathway in a mammalian cell. Preferably the mammalian cellis a cancer cell.

In a preferred embodiment, the dominant negative MINK3 protein willinhibit growth factor-induced ERK activation in a mammalian cell,preferably a cancer cell.

In a preferred embodiment, the dominant negative MINK3 protein willinhibit growth factor-dependent proliferation in a mammalian cell.

In another preferred embodiment, the dominant negative MINK3 proteinwill inhibit cell cycle progression and proliferation in a mammaliancell. Preferably the mammalian cell is a cancer cell and/or alymphocyte.

In a preferred embodiment, the dominant negative MINK3 protein is akinase dead MINK3 protein variant as described herein.

In a preferred embodiment, the dominant negative kinase dead MINK3protein variant has a mutation in an ATP-binding domain. Preferably thenon-mutant ATP-binding domain of the non-variant MINK3 protein(non-variant with respect to ATP-binding domain) comprises the aminoacid sequence set forth by amino acids 32-54 in SEQ ID NO:2. In apreferred embodiment, the dominant negative kinase dead MINK3 proteinvariant has a substitution mutation in the ATP binding domain at aposition corresponding to K54 in SEQ ID NO:2.

In one aspect, the invention is directed to methods for screeningcandidate bioactive agents for an ability to bind to MINK3 proteins. Ina preferred embodiment, the methods comprise combining a MINK3 proteinand a candidate bioactive agent and determining the binding of candidatebioactive agent to MINK3 protein.

In another aspect, the invention is directed to methods for screening acandidate bioactive agent for an ability to interfere with the bindingof a MINK3 protein. In one embodiment, the interference is between thebinding of anti-MINK3 antibody and MINK3 protein. In a preferredembodiment, the interference is between the binding of a MINK3 proteinand an Nck protein such as an Nck protein comprising the amino acidsequence set forth at Genbank accession number MD13752. In oneembodiment, such a method comprises combining a MINK3 protein, acandidate bioactive agent and an Nck protein, and determining thebinding of the MINK3 protein to Nck protein in the presence of candidatebioactive agent. Preferably, the binding of MINK3 to Nck is determinedin the presence and absence of candidate bioactive agent. If desired,the MINK3 protein and the Nck protein can be combined first.

In another aspect, the invention is directed to methods for screeningcandidate bioactive agents for an ability to modulate MINK3 proteinactivity. In a preferred embodiment, the methods comprise combining aMINK3 protein and a candidate bioactive agent and determining the effectof the candidate agent on the activity of MINK3 protein. In a preferredembodiment, a library of candidate bioactive agents is added to aplurality of cells comprising a recombinant nucleic acid encoding aMINK3 protein and MINK3 protein activity is determined. Preferably,MINK3 protein activity is determined in the presence and absence ofcandidate bioactive agent.

In one aspect, the invention is directed to methods for screening for abioactive agent capable of modulating JNK phosphorylation and/oractivation. In one aspect, the invention is directed to methods forscreening for a bioactive agent capable of modulating the JNK signaltransduction pathway. In a preferred embodiment, the methods comprisecontacting a candidate bioactive agent to a mammalian cell comprising arecombinant MINK3 nucleic acid encoding a MINK3 protein and a JNKprotein and determining JNK activity in the presence of candidate agent.In a preferred embodiment, JNK activity is determined in the presenceand absence of candidate agent. The recombinant MINK3 nucleic acid isexpressed in said mammalian cell and will activate JNK protein in theabsence of candidate bioactive agent. In a preferred embodiment, theencoded MINK3 protein comprises an amino acid sequence having at leastabout 90% identity to an amino acid sequence selected from the groupconsisting of the amino acid sequences set forth in SEQ ID NOs: 2, 4 and6. A decrease in the activity of JNK protein in the presence ofcandidate bioactive agent indicates that the candidate bioactive agentis capable of modulating JNK activity.

In one aspect, the invention is directed to methods for screening for abioactive agent capable of modulating ERK phosphorylation and/oractivation. In one aspect, the invention is directed to methods forscreening for a bioactive agent capable of modulating the ERK signaltransduction pathway. In a preferred embodiment, the methods comprisecontacting a candidate bioactive agent to a mammalian cell comprising arecombinant MINK3 nucleic acid encoding a MINK3 protein and a ERKprotein and determining ERK activity in the presence of candidate agent.In a preferred embodiment, ERK activity is determined in the presenceand absence of candidate agent. The recombinant MINK3 nucleic acid isexpressed in said mammalian cell and will activate ERK protein in theabsence of candidate bioactive agent. In a preferred embodiment, theencoded MINK3 protein comprises an amino acid sequence having at leastabout 90% identity to an amino acid sequence selected from the groupconsisting of the amino acid sequences set forth in SEQ ID NOs: 2, 4 and6. A decrease in the activity of ERK protein in the presence ofcandidate bioactive agent indicates that the candidate bioactive agentis capable of modulating ERK activity.

In a preferred embodiment, the methods comprise contacting a mammaliancell with a growth factor which will activate JNK and/or ERK. In apreferred embodiment, the growth factor used in epidermal growth factor(EGF).

In one aspect, the invention is directed to methods for screeningcandidate bioactive agents for an ability to modulate proliferation,survival, migration, morphology, cytoskeletal organization andintracellular signal transduction in mammalian cells. Such cells arepreferably cancer cells and/or lymphocytes. In a preferred embodiment,the method involves screening for a bioactive agent capable of bindingto MINK3 protein using assays provided herein. In another preferredembodiment, the method involves screening for a bioactive agent capableof modulating MINK3 binding using assays provided herein. In anotherpreferred embodiment, the method involves screening for a bioactiveagent capable of modulating MINK3 activity using assays provided herein.

In a preferred embodiment the methods comprise combining a MINK3protein, a candidate bioactive agent and a cell or a population of cellsand determining the effect on the cell in the presence and absence ofcandidate agent.

In a preferred embodiment, the methods comprise introducing arecombinant MINK3 nucleic acid into a host cell capable of expressingthe nucleic acid, contacting the cell with a candidate bioactive agent,and determining the effect on the cell in the presence and absence ofcandidate bioactive agent. In another preferred embodiment, a library ofcandidate bioactive agents is added to a plurality of cells comprising arecombinant nucleic acid encoding a MINK3 protein.

A MINK3 protein used in screening methods provided herein may berecombinant, isolated or cell-free as in a cell lysate.

Preferred candidate bioactive agents for use in screening methodsprovided herein include small molecule chemical compounds.

In another aspect, the invention provides compositions and methods fordiagnostic and prognostic determination of disorders involving MINK3dysfunction and/or dysregulation. Without being bound by theory, suchdisorders involve the dysregulation of MINK3 gene expression, aberrantMINK3 gene structure and/or modification, the dysregulation and/ordysfunction of MINK3 protein, and aberrant MINK3 protein structureand/or modification.

Further provided herein are compositions and methods for prophylaxis andtherapeutic treatment of disorders related to and/or involving MINK3dysfunction or dysregulation. In a preferred embodiment, such disordersinclude cancer. In a preferred embodiment, such disorders involvedysfunction or dysregulation of leukocyte function, preferablylymphocyte function.

In one aspect, the present invention provides isolated polypeptideswhich specifically bind to a MINK3 protein as described herein. In apreferred embodiment, the isolated peptide is an anti-MINK3 antibody. Ina further preferred embodiment, the isolated peptide is an anti-MINK3monoclonal antibody. In a preferred embodiment, the anti-MINK3 antibodywill reduce or eliminate the biological function of MINK3 protein.

In a preferred embodiment, the present invention provides MINK3 proteinsand nucleic acids, as well as agents that bind to them and/or modulatetheir activity, including and preferably small molecule chemicalcompositions as discussed herein, which are useful in the treatment ofacute and chronic inflammatory diseases and autoimmune diseases, as wellas in the treatment of a host receiving a transplant, as well asdiseases characterized by immunodeficiency.

The dysregulation of mechanisms of programmed cell death can lead tocancer, particularly in lymphocytes (Chao et al., Ann. Rev. Immunol.16:395419, 1998). For example, overexpression of Bcl-2, which isinvolved in normal cell survival through the inhibition of apoptosis, isthought to be responsible for the survival of excessive numbers oflymphocytes in a form of lymphoma.

Without being bound by theory, the present invention provides MINK3proteins and nucleic acids, as well as agents that bind to them and/ormodulate their activity, including and preferably small moleculechemical compositions as discussed herein, which are useful in themodulation of T cell and B cell survival and apoptosis.

Other aspects of the invention will become apparent to the skilledartisan by the following description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleic acid sequence and amino acid sequence ofMINK3a, MINK3b, and MINK3c. SEQ ID NOs: 1 and 2 depict the nucleic acidand amino acid sequences of MINK3a, respectively. SEQ ID NOs: 3 and 4depict the nucleic acid and amino acid sequences of MINK3b,respectively. SEQ ID NOs: 5 and 6 depict the nucleic acid and amino acidsequences of MINK3c, respectively.

FIG. 2 schematically describes the structure of MINK1 and MINK3 isoformsa and b. The kinase domain, intermediate region, and GCK (GCKH) domainare identified, as is the PxxPxR motif which interacts with the SH3domain of Nck. The relative position of a MINK3 antisense nucleic acid(“MINK3 as (TR5)”) is also shown. It is recognized that the MINK3antisense nucleic acid described is directed to MINK1 as well. It isalso recognized that MINK3b and MINK3c have a frameshift relative toMINK3a; consequently MINK3b and MINK3c proteins comprising MINK3b andMINK3c amino acid sequence are kinase dead MINK3 proteins.

MINK3 antisense nucleic acid is herein equivalently referred to asantisense MINK3 nucleic acid, antisense MINK nucleic acid, MINKantisense, MINK antisense nucleic acid, and grammatical equivalentsthereof.

FIG. 3 graphically depicts the effects of MINK3a antisense nucleic acidon AP-1 modulated transcriptional activity in 293 cells. MEKK1 means MEKkinase-1.

FIG. 4 schematically describes signal transduction pathways mediatingthe response of mammalian cells to taxol.

FIGS. 5 and 6 schematically describes the signal transduction pathwaysthat propogate signals initiated by cytokines, mitogens, and cellularstress.

FIG. 7 graphically demonstrates the ability of MINK3 antisense nucleicacid to inhibit taxol-induced cell death in Hela cells.

FIG. 8 graphically demonstrates the ability of MINK3a to inhibitproliferation of the human tumor cell line A549 in low serum.

FIG. 9 graphically demonstrates the ability of MINK3 antisense nucleicacid to inhibit EGF-induced ERK-mediated transcriptional activation.

FIG. 10 shows Western blots for retinoblastoma “Rb” and cleaved Rb andBcl-2 protein from cells expressing (1) Bcl2; (2) MINK3 antisensenucleic acid “TR5”; (3) GFP;and (4), (5) cells transfected with emptyvector; in the presence and absence of taxol.

FIG. 11 shows Northern blot of MINK3 mRNA in human tissue samples.

FIG. 12 shows Northern blot of MINK3 mRNA in tumor cell lines.

FIG. 13 shows JNK kinase assay using GST-cJUN as substrate, and ERKkinase assay using MBP as substrate, done on extracts from cellstransfected with empty vector “pCDNA”; MINK3a, MINK3b or MEKK1. Westernblots “WB” are also done for JNK and ERK proteins.

FIG. 14 shows MDA-MB-231 human tumor cells expressing recombinantMINK3a, MINK3b, antisense MINK nucleic acid, or GFP.

FIG. 15 shows stably transfected MDA-MB-231 cells expressing recombinantMINK3a “MINK3a”, and MDA-MB-231 cells alone “MDA-MB-231”, and stablytransfected MDA-MB-231 cells expressing recombinant MINK3a treated withthe MEK inhibitor PD98059.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for modulatingproliferation, survival, migration, morphology, cytoskeletalorganization and intracellular signal transduction in mammalian cells.Nucleic acids encoding proteins and proteins so encoded which arecapable of modulating proliferation, survival, migration, morphology,cytoskeletal organization and intracellular signal transduction inmammalian cells are provided. Compositions and methods for the treatmentof disorders related to cell proliferation, survival, morphology andmigration are also provided. Prophylactics and methods for theprevention of such disorders are also provided. Also provided arecompositions and methods for diagnostic and prognostic determination ofsuch disorders. Further provided are assays for the identification ofbioactive agents capable of modulating signal transduction,proliferation, survival, morphology and migration in mammalian cells.

The present invention provides MINK3 nucleic acids, including nucleicacids encoding MINK3 protein, which are capable of modulatingproliferation, survival, migration, morphology, cytoskeletalorganization and intracellular signal transduction in mammalian cells.Also provided herein are MINK3 antisense nucleic acids which are capableof modulating proliferation, survival, migration, morphology,cytoskeletal organization and intracellular signal transduction inmammalian cells. Also provided herein are MINK3 proteins, includingdominant negative MINK3 proteins, which are capable of modulatingproliferation, survival, migration, morphology, cytoskeletalorganization and intracellular signal transduction in mammalian cells.

MINK3 proteins of the present invention include proteins, polypeptides,and peptides. Among the MINK3 proteins included herein are dominantnegative isoforms of MINK3 proteins which will inhibit the activity ofnon-mutant MINK3 proteins in the presence thereof.

In one embodiment, a MINK3 protein has one or more of the followingcharacteristics (MINK3 bioactivities): binding to Nck (for example, anNck protein comprising the amino acid sequence set forth at Genbankaccession no. AAD13752); kinase activity directed at JNK, preferablyJNK2; kinase activity directed at ERK, preferably ERK1; an ability toactivate JNK and/or ERK, preferably JNK2 and ERK1; an ability toactivate JNK and/or ERK signal transduction pathways in mammalian cells;an ability to effect a change in cell morphology in mammalian cells,preferably cancer cells; an ability to disrupt F-actin in mammaliancells, preferably cancer cells.

In a preferred embodiment, MINK3 protein binds to Nck protein via thePxxPxR amino acid motif in MINK3 protein. In a preferred embodiment,MINK3 protein binds to Nck in mammalian cells. IN one embodiment, MINK3binds to Nck in tumor cells, for example 293 cells.

Also provided herein are MINK3 dominant negative proteins which inhibitat least one MINK3 protein activity. In a preferred embodiment, theMINK3 dominant negative protein has one or more of the followingcharacteristics (MINK3 dominant negative activities): an ability to bindNck protein; an inability to bind Nck protein; an ability to inhibitgrowth factor-induced ERK activation, preferably EGF-induced ERKactivation; an ability to inhibit proliferation in a mammalian cell,preferably a cancer cell; an ability to inhibit growth factor-inducedproliferation in a mammalian cell, preferably EGF-induced proliferation;an ability to inhibit growth factor-dependent proliferation in amammalian cell, preferably EGF-dependent proliferation; an ability toinhibit aberrant cell proliferation, preferably involving aberrant JNKand/or ERK activation, preferably in cancer cells; an ability to inhibitphosphorylation of c-JUN N-terminal kinase (JNK) and/or ERK, preferablyJNK2 and/or ERK1; an ability to inhibit activation of JNK and/or ERK,preferably JNK2 and/or ERK1; an ability to inhibit the JNK signaltransduction pathway and/or the ERK signal transduction pathway in amammalian cell, preferably a cancer cell, preferably a lymphocyte; anability to inhibit taxol-induced cleavage of Rb and apoptosis in amammalian cell; an ability to promote survival in a mammalian cellfollowing exposure to taxol; an ability to inhibit the transcriptionpromoting activity of AP-1 in a mammalian cell; an ability to inhibittranscriptional activation by one or more AP-1 response elements.

In a preferred embodiment, the MINK3 protein has one or more of theMINK3 bioactivities described herein. In other embodiments whereparticular bioactivities are not required, a MINK3 protein does notinclude all bioactivities described herein for MINK3 proteins.

It has been reported that the adaptor protein Nck links receptortyrosine kinases with the serine-threonine kinase Pak1. Nck is anadaptor protein composed of a single SH2 domain and three SH3 domains.Upon growth factor stimulation, Nck is recruited to receptor tyrosinekinases via its SH2 domain, probably initiating one or more signalingcascades. Galisteo, et al., J Biol Chem. 271(35):20997-1000 (1996). Alsosee, Chen, et al., J Biol Chem., 273(39):25171-8 (1998) which reports onNck family genes, chromosomal localization and expression.

In one embodiment, MINK3 nucleic acids or MINK3 proteins are initiallyidentified by substantial nucleic acid and/or amino acid sequenceidentity or similarity to sequences provided herein. In a preferredembodiment, MINK3 nucleic acids or MINK3 proteins have sequence identityor similarity to the sequences provided herein as described below andone or more of the MINK3 protein bioactivities (or MINK3 dominantnegative activities or MINK3 antisense nucleic acid activities) asdescribed herein. Such sequence identity or similarity can be based uponthe overall nucleic acid or amino acid sequence.

In a preferred embodiment, a protein is a “MINK3 protein” as definedherein if it comprises an amino acid sequence having at least about 80%,more preferably at least about 85%,more preferably at least about 90%,more preferably at least about 95%, more preferably at least about 98%identity to an amino acid sequence selected from the group consisting ofthe amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6.

In a preferred embodiment, the MINK3 protein comprises an amino acidsequence selected from the group consisting of the amino acid sequencesset forth in SEQ ID NOs: 2, 4, and 6.

In another preferred embodiment, a MINK3 protein has an overall sequencesimilarity to an amino acid sequence selected from the group consistingof the amino acid sequences set forth in SEQ ID NOs: 2, 4, and 6 ofgreater than at least about 80%, more preferably at least about 85%,more preferably at least about 90%, more preferably at least about 95%,more preferably at least about 98%, more preferably 100%.

In another preferred embodiment, a MINK 3 protein provided hereincomprises a GCK domain comprising an amino acid sequence having at leastabout 80%, more preferably at least about 85%, more preferably at leastabout 90%, more preferably at least about 95%, more preferably at leastabout 98%, more preferably about 99%, more preferably 100% identity tothe amino acid sequence set forth by amino acids 994-1292 or 994-1290 inSEQ ID NO:2.

In another preferred embodiment, a MINK 3 protein provided hereincomprises a catalytic serine/threonine kinase domain comprising an aminoacid sequence having at least about 80%, more preferably at least about85%, more preferably at least about 90%, more preferably at least about95%, more preferably at least about 98%, more preferably about 99%, morepreferably 100% identity to the amino acid sequence set forth by aminoacids 25-289 in SEQ ID NO:2.

In another preferred embodiment, a MINK 3 protein provided hereincomprises a catalytic tyrosine kinase domain comprising an amino acidsequence having at least about 80%, more preferably at least about 85%,more preferably at least about 90%, more preferably at least about 95%,more preferably at least about 98%, more preferably about 99%, morepreferably 100% identity to the amino acid sequence set forth by aminoacids 26-286 in SEQ ID NO:2.

In another preferred embodiment, a MINK 3 protein provided hereincomprises an ATP binding domain comprising an amino acid sequence havingat least about 80%, more preferably at least about 85%, more preferablyat least about 90%, more preferably at least about 95%, more preferablyat least about 98%, more preferably about 99%, more preferably 100%identity to the amino acid sequence set forth by amino acids 32-54 inSEQ ID NO:2.

As is known in the art, a number of different programs can be used toidentify whether a protein (or nucleic acid as discussed below) hassequence identity or similarity to a known sequence. Sequence identityand/or similarity is determined using standard techniques known in theart, including, but not limited to, the local sequence identityalgorithm of Smith, et al., Adv. Appl. Math. 2:482 (1981), by thesequence identity alignment algorithm of Needleman, et al., J. Mol.Biol., 48:443 (1970), by the search for similarity method of Pearson, etal., PNAS USA, 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux, et al.,Nucl. Acid Res., 12:387-395 (1984), preferably using the defaultsettings, or by inspection. Preferably, percent identity is calculatedby FastDB based upon the following parameters: mismatch penalty of 1;gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30,“Current Methods in Sequence Comparison and Analysis,” MacromoleculeSequencing and Synthesis, Selected Methods and Applications, pp 127-149(1988), Alan R. Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng, et al., J. Mol. Evol.,35:351-360 (1987); the method is similar to that described by Higgins,et al., CABIOS, 5:151-153 (1989). Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul, et al., J. Mol. Biol,. 215:403410, (1990) and Karlin, etal., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST programis the WU-BLAST-2 program which was obtained from Altschul, et al.,Methods in Enzymology, 266:460-480 (1996);http://blast.wustl/edu/blast/README.html]. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0. 125, word threshold (T)=11. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschul,et al., Nucleic Acids Res., 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions; charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to ˜22 bits.

A % amino acid sequence identity value is determined by the number ofmatching identical residues divided by the total number of residues ofthe “longer” sequence in the aligned region. The “longer” sequence isthe one having the most actual residues in the aligned region (gapsintroduced by WU-Blast-2 to maximize the alignment score are ignored).

In a similar manner, “percent (%) nucleic acid sequence identity” withrespect to the coding sequence of the polypeptides identified herein isdefined as the percentage of nucleotide residues in a candidate sequencethat are identical with the nucleotide residues in the coding sequenceof the MINK3 protein. A preferred method utilizes the BLASTN module ofWU-BLAST-2 set to the default parameters, with overlap span and overlapfraction set to 1 and 0.125, respectively.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than a protein comprising an amino acid sequenceselected from the group consisting of the amino acid sequences set forthin SEQ ID NOs: 2, 4, and 6, it is understood that in one embodiment, thepercentage of sequence identity will be determined based on the numberof identical amino acids in relation to the total number of amino acids.Thus, for example, sequence identity of sequences shorter than thatshown in SEQ ID NO:2. 4. or 6, as discussed below, will be determinedusing the number of amino acids in the shorter sequence, in oneembodiment. In percent identity calculations relative weight is notassigned to various manifestations of sequence variation, such as,insertions, deletions, substitutions, etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

As will be appreciated by those skilled in the art, the sequences of thepresent invention may contain sequencing errors. That is, there may beincorrect nucleosides, frameshifts, unknown nucleosides, or other typesof sequencing errors in any of the sequences; however, the correctsequences will fall within the homology and stringency definitionsherein.

MINK3 proteins of the present invention may be shorter or longer thanthe amino acid sequences encoded by the nucleic acid sequences set forthin SEQ ID NOs:1, 3, and 5. Thus, in a preferred embodiment, includedwithin the definition of MINK3 proteins are portions or fragments of theamino acid sequences encoded by the nucleic acid sequences providedherein. In one embodiment herein, fragments of MINK3 proteins areconsidered MINK3 proteins if a) they share at least one antigenicepitope; b) have at least the indicated sequence identity; c) andpreferably have MINK3 biological activity as further defined herein. Insome cases, where the sequence is used diagnostically, that is, when thepresence or absence of MINK3 nucleic acid is determined, only theindicated sequence identity is required. The nucleic acids of thepresent invention may also be shorter or longer than the sequences setforth in SEQ ID NOs:1, 3, and 5. The nucleic acid fragments include anyportion of the nucleic acids provided herein which have a sequence notexactly previously identified; fragments having sequences with theindicated sequence identity to that portion not previously identifiedare provided in an embodiment herein.

In addition, as is more fully outlined below, MINK3 proteins can be madethat are longer than those consisting essentially of an amino acidsequence selected from the group consisting of the amino acid sequencesset forth in SEQ ID NOs:2, 4, and 6; for example, by the addition ofepitope or purification tags, the addition of other fusion sequences, orthe elucidation of additional coding and non-coding sequences. Asdescribed below, the fusion of a MINK3 peptide to a fluorescent peptide,such as Green Fluorescent Peptide (GFP), is particularly preferred.

MINK3 proteins may also be identified as encoded by MINK3 nucleic acidswhich hybridize to a nucleic acid comprising a nucleic acid sequenceselected from the group consisting of the nucleic acid sequences setforth in SEQ ID NOs:1, 3, and 5. Hybridization conditions are furtherdescribed below.

In a preferred embodiment, when a MINK3 protein is to be used togenerate antibodies, a MINK3 protein must share at least one epitope ordeterminant with the full length protein. By “epitope” or “determinant”herein is meant a portion of a protein which will generate and/or bindan antibody. Thus, in most instances, antibodies made to a smaller MINK3protein will be able to bind to the full length protein. In a preferredembodiment, the epitope is unique; that is, antibodies generated to aunique epitope show little or no cross-reactivity. The term “antibody”includes antibody fragments, as are known in the art, including FabFab₂, single chain antibodies (Fv for example), chimeric antibodies,etc., either produced by the modification of whole antibodies or thosesynthesized de novo using recombinant DNA technologies.

In a preferred embodiment, the antibodies to a MINK3 protein are capableof reducing or eliminating the biological function of the MINK3 proteinsdescribed herein, as is described below. That is, the addition ofanti-MINK3 protein antibodies (either polyclonal or preferablymonoclonal) to MINK3 proteins (or cells containing MINK3 proteins) mayreduce or eliminateone or more MINK3 bioactivities as described herein.Generally, at least a 25% decrease in activity is preferred, with atleast about 50% being particularly preferred and about a 95-100%decrease being especially preferred.

The MINK3 antibodies of the invention specifically bind to MINK3proteins. By “specifically bind” herein is meant that the antibodiesbind to the protein with a binding constant in the range of at least10⁻⁴-10⁻⁶ M⁻¹, with a preferred range being 10⁻⁷-10⁻⁹ M⁻¹. Antibodiesare further described below.

In the case of the nucleic acid, the overall sequence identity of thenucleic acid sequence is commensurate with amino acid sequence identitybut takes into account the degeneracy in the genetic code and codon biasof different organisms. Accordingly, the nucleic acid sequence identitymay be either lower or higher than that of the protein sequence. Thusthe sequence identity of the nucleic acid sequence as compared to anucleic acid sequence selected from the group of nucleic acid sequencesset forth in SEQ ID NOs:1, 3, and 5 is preferably greater than about80%, more preferably greater than about 85%, more preferably greaterthan about 90%, more preferably greater than about 95%, more preferablygreater than about 98%, more preferably about 99%, more preferably 100%.

In a preferred embodiment, a MINK3 nucleic acid encodes a MINK3 protein.As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids may be made,all of which encode the MINK3 proteins of the present invention. Thus,having identified a particular amino acid sequence, those skilled in theart could make any number of different nucleic acids, by simplymodifying the sequence of one or more codons in a way which does notchange the amino acid sequence of the MINK3 protein.

In one embodiment, the nucleic acid is determined through hybridizationstudies. Thus, for example, nucleic acids which hybridize under highstringency conditions to a nucleic acid comprising a nucleic acidsequence selected from the group consisting of the nucleic acidsequences set forth in SEQ ID NOs:1, 3, and 5, or complements thereof,are considered MINK3 nucleic acids. High stringency conditions,including washing conditions, are known in the art; see for exampleManiatis, et al., Molecular Cloning: A Laboratory Manual, 2d Edition,1989, and Current Protocols in Molecular Biology, eds. F. Ausubel etal., New York, Greene Pub. Associates & Wiley Interscience, 1988; bothof which are hereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength pH. The T_(m) is the temperature (under defined ionic strength,pH and nucleic acid concentration) at which 50% of the probescomplementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g. greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. Wash conditions for stringent hybridizationtypically involve a lower sodium (or other salt) concentration, and alower temperature, than that used in the hybridization step, asdiscussed in Maniatis (supra), Ausubel (supra) and Tijssen (supra).

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions may be used, as areknown in the art; see Maniatis and Ausubel, (supra), and Tijssen(supra).

The MINK3 proteins and nucleic acids of the present invention arepreferably recombinant. As used herein and further defined below,“nucleic acid” may refer to either DNA or RNA, or molecules whichcontain both deoxy- and ribonucleotides. The nucleic acids includegenomic DNA, cDNA and oligonucleotides including sense and anti-sensenucleic acids. Such nucleic acids may also contain modifications in theribose-phosphate backbone to increase stability and half life of suchmolecules in physiological environments.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. As will beappreciated by those in the art, the depiction of a single strand(“Watson”) also defines the sequence of the other strand (“Crick”); thusthe sequences depicted in SEQ ID NOs:1, 3, and 5 also includecomplements thereof. By the term “recombinant nucleic acid” herein ismeant nucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid by endonucleases, in a form not normallyfound in nature. Thus an isolated MINK3 nucleic acid, in a linear form,or an expression vector formed in vitro by ligating DNA molecules thatare not normally joined, are both considered recombinant for thepurposes of this invention. It is understood that once a recombinantnucleic acid is made and reintroduced into a host cell or organism, itwill replicate non-recombinantly, i.e. using the in vivo cellularmachinery of the host cell rather than in vitro manipulations; however,such nucleic acids, once produced recombinantly, although subsequentlyreplicated non-recombinantly, are still considered recombinant for thepurposes of the invention.

Similarly, a “recombinant protein” is a protein made using recombinanttechniques, i.e. through the expression of a recombinant nucleic acid asdepicted above. A recombinant protein is distinguished from naturallyoccurring protein by at least one or more characteristics. For example,the protein may be isolated or purified away from some or all of theproteins and compounds with which it is normally associated in its wildtype host, and thus may be substantially pure. For example, an isolatedprotein is unaccompanied by at least some of the material with which itis normally associated in its natural state, preferably constituting atleast about 0.5%, more preferably at least about 5% by weight of thetotal protein in a given sample. A substantially pure protein comprisesat least about 75% by weight of the total protein, with at least about80% being preferred, and at least about 90% being particularlypreferred. The definition includes the production of a MINK3 proteinfrom one organism in a different organism or host cell. Alternatively,the protein may be made at a significantly higher concentration than isnormally seen, through the use of a inducible promoter or highexpression promoter, such that the protein is made at increasedconcentration levels. Alternatively, the protein may be in a form notnormally found in nature, as in the addition of an epitope tag or aminoacid substitutions, insertions and deletions, as discussed below.

In one embodiment, the present invention provides MINK3 proteinvariants. These variants fall into one or more of three classes:substitutional, insertional or deletional variants. These variantsordinarily are prepared by site specific mutagenesis of nucleotides inthe DNA encoding a MINK3 protein, using cassette or PCR mutagenesis orother techniques well known in the art, to produce DNA encoding thevariant, and thereafter expressing the DNA in recombinant cell cultureas outlined above. However, variant MINK3 protein fragments having up toabout 100-150 residues may be prepared by in vitro synthesis usingestablished techniques. Amino acid sequence variants are characterizedby the predetermined nature of the variation, a feature that sets themapart from naturally occurring allelic or interspecies variation of theMINK3 protein amino acid sequence. The variants typically exhibit thesame qualitative biological activity as the naturally occurringanalogue, although variants can also be selected which have modifiedcharacteristics as will be more fully outlined below.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed MINK3 variants screened for theoptimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants is done using assays of MINK3protein activities.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the MINK3 protein aredesired, substitutions are generally made in accordance with thefollowing chart:

CHART I Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, LeuSubstantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the MINK3 proteins as needed. Alternatively, thevariant may be designed such that the biological activity of the MINK3protein is altered. For example, glycosylation sites may be altered orremoved.

In one aspect the invention provides dominant negative MINK3 proteinswhich inhibit at least one MINK3 protein activity, as described herein.In a [referred embodiment, the dominant negative MINK3 protein is akinase dead MINK3 protein. In a preferred embodiment, the dominantnegative kinase dead MINK3 protein variant has a mutation in anATP-binding domain. Preferably the non-mutant ATP-binding domain of thenon-variant MINK3 protein (non-variant with respect to ATP-bindingdomain) comprises the amino acid sequence set forth by amino acids 32-54in SEQ ID NO:2. In a preferred embodiment, the dominant negative kinasedead MINK3 protein variant has a substitution mutation in the ATPbinding domain at a position corresponding to K54 in SEQ ID NO:2.

In a preferred embodiment, the dominant negative MINK3 protein has MINK3antisense nucleic acid activity.

Covalent modifications of MINK3 polypeptides are included within thescope of this invention. One type of covalent modification includesreacting targeted amino acid residues of a MINK3 polypeptide with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N-or C-terminal residues of a MINK3 polypeptide.Derivatization with bifunctional agents is useful, for instance, forcrosslinking MINK3 to a water-insoluble support matrix or surface foruse in the method for purifying anti-MINK3 antibodies or screeningassays, as is more fully described below. Commonly used crosslinkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidyl-propionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of the“-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the MINK3 polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence MINK3polypeptide, and/or adding one or more glycosylation sites that are notpresent in the native sequence MINK3 polypeptide.

Addition of glycosylation sites to MINK3 polypeptides may beaccomplished by altering the amino acid sequence thereof. The alterationmay be made, for example, by the addition of, or substitution by, one ormore serine or threonine residues to the native sequence MINK3polypeptide (for O-linked glycosylation sites). The MINK3 amino acidsequence may optionally be altered through changes at the DNA level,particularly by mutating the DNA encoding the MINK3 polypeptide atpreselected bases such that codons are generated that will translateinto the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theMINK3 polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the MINK3 polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge, et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo-and exo-glycosidases asdescribed by Thotakura, et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification comprises linking the MINK3polypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

MINK3 polypeptides of the present invention may also be modified in away to form chimeric molecules comprising a MINK3 polypeptide fused toanother, heterologous polypeptide or amino acid sequence. In oneembodiment, such a chimeric molecule comprises a fusion of a MINK3polypeptide with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino-or carboxyl-terminus of the MINK3 polypeptide. Thepresence of such epitope-tagged forms of a MINK3 polypeptide can bedetected using an antibody against the tag polypeptide. Also, provisionof the epitope tag enables the MINK3 polypeptide to be readily purifiedby affinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. In an alternativeembodiment, the chimeric molecule may comprise a fusion of a MINK3polypeptide with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule, such afusion could be to the Fc region of an IgG molecule as discussed furtherbelow.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field, et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan, et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky, et al., Protein Engineering,3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide[Hopp, et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitopepeptide [Martin, et al., Science 255:192-194 (1992)]; tubulin epitopepeptide [Skinner, et al., J. Biol. Chem., 266:15163-15166 (1991)]; andthe T7 gene 10 protein peptide tag [Lutz-Freyermuth, et al., Proc. Natl.Acad. Sci. USA, 87:6393-6397 (1990)].

In an embodiment herein, MINK3 genes from other organisms are cloned andexpressed as outlined below. Thus, probe or degenerate polymerase chainreaction (PCR) primer sequences may be used to find other related MINK3proteins from humans or other organisms. As will be appreciated by thosein the art, particularly useful probe and/or PCR primer sequencesinclude the unique areas of the MINK3 nucleic acid sequence. As isgenerally known in the art, preferred PCR primers are from about 15 toabout 35 nucleotides in length, with from about 20 to about 30 beingpreferred, and may contain inosine as needed. The conditions for the PCRreaction are well known in the art. It is therefore also understood thatprovided along with the sequences in the sequences listed herein areportions of those sequences, wherein unique portions of 15 nucleotidesor more are particularly preferred. The skilled artisan can routinelysynthesize or cut a nucleotide sequence to the desired length.

Once isolated from its natural source, e.g., contained within a plasmidor other vector or excised therefrom as a linear nucleic acid segment,the recombinant MINK3 nucleic acid can be further-used as a probe toidentify and isolate other MINK3 nucleic acids. It can also be used as a“precursor” nucleic acid to make modified or variant MINK3 nucleic acidsand proteins.

Using the nucleic acids of the present invention which encode a MINK3protein, a variety of expression vectors are made. The expressionvectors may be either self-replicating extrachromosomal vectors orvectors which integrate into a host genome. Generally, these expressionvectors include transcriptional and translational regulatory nucleicacid operably linked to the nucleic acid encoding the MINK3 protein. Theterm “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. As another example, operablylinked refers to DNA sequences linked so as to be contiguous, and, inthe case of a secretory leader, contiguous and in reading phase.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice. The transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the MINK3 protein; for example, transcriptional andtranslational regulatory nucleic acid sequences from Bacillus arepreferably used to express the MINK3 protein in Bacillus. Numerous typesof appropriate expression vectors, and suitable regulatory sequences areknown in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. In apreferred embodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammalianor insect cells for expression and in a procaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

A preferred expression vector system is a retroviral vector system suchas is generally described in PCT/US97/01019 and PCT/US97/01048, both ofwhich are hereby expressly incorporated by reference.

MINK3 proteins of the present invention are produced by culturing a hostcell transformed with an expression vector containing nucleic acidencoding a MINK3 protein, under the appropriate conditions to induce orcause expression of the MINK3 protein. The conditions appropriate forMINK3 protein expression will vary with the choice of the expressionvector and the host cell, and will be easily ascertained by one skilledin the art through routine experimentation. For example, the use ofconstitutive promoters in the expression vector will require optimizingthe growth and proliferation of the host cell, while the use of aninducible promoter requires the appropriate growth conditions forinduction. In addition, in some embodiments, the timing of the harvestis important. For example, the baculoviral systems used in insect cellexpression are lytic viruses, and thus harvest time selection can becrucial for product yield.

Appropriate host cells include yeast, bacteria, archebacteria, fungi,and insect and animal cells, including mammalian cells. Of particularinterest are Drosophila melanogaster cells, Saccharomyces cerevisiae andother yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanomacell lines, immortalized mammalian myeloid and lymphoid cell lines, andhuman tumor lines, including and preferably MDA-MB-231 cells.

In a preferred embodiment, the MINK3 proteins are expressed in mammaliancells. Mammalian expression systems are also known in the art, andinclude retroviral systems. A mammalian promoter is any DNA sequencecapable of binding mammalian RNA polymerase and initiating thedownstream (3′) transcription of a coding sequence for MINK3 proteininto mRNA. A promoter will have a transcription initiating region, whichis usually placed proximal to the 5′ end of the coding sequence, and aTATA box, using a located 25-30 base pairs upstream of the transcriptioninitiation site. The TATA box is thought to direct RNA polymerase II tobegin RNA synthesis at the correct site. A mammalian promoter will alsocontain an upstream promoter element (enhancer element), typicallylocated within 100 to 200 base pairs upstream of the TATA box. Anupstream promoter element determines the rate at which transcription isinitiated and can act in either orientation. Of particular use asmammalian promoters are the promoters from mammalian viral genes, sincethe viral genes are often highly expressed and have a broad host range.Examples include the SV40 early promoter, mouse mammary tumor virus LTRpromoter, adenovirus major late promoter, herpes simplex virus promoter,and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenlytion signals includethose derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, viral infection, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

In a preferred embodiment, MINK3 proteins are expressed in bacterialsystems. Bacterial expression systems are well known in the art.

A suitable bacterial promoter is any nucleic acid sequence capable ofbinding bacterial RNA polymerase and initiating the downstream (3′)transcription of the coding sequence of MINK3 protein into mRNA. Abacterial promoter has a transcription initiation region which isusually placed proximal to the 5′ end of the coding sequence. Thistranscription initiation region typically includes an RNA polymerasebinding site and a transcription initiation site. Sequences encodingmetabolic pathway enzymes provide particularly useful promotersequences. Examples include promoter sequences derived from sugarmetabolizing enzymes, such as galactose, lactose and maltose, andsequences derived from biosynthetic enzymes such as tryptophan.Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;for example, the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosomebinding site is desirable. In E. coli, the ribosome binding site iscalled the Shine-Delgarno (SD) sequence and includes an initiation codonand a sequence 3-9 nucleotides in length located 3-11 nucleotidesupstream of the initiation codon.

The expression vector may also include a signal peptide sequence thatprovides for secretion of the MINK3 protein in bacteria. The signalsequence typically encodes a signal peptide comprised of hydrophobicamino acids which direct the secretion of the protein from the cell, asis well known in the art. The protein is either secreted into the growthmedia (gram-positive bacteria) or into the periplasmic space, locatedbetween the inner and outer membrane of the cell (gram-negativebacteria).

The bacterial expression vector may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed. Suitable selection genes include genes which render thebacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways.

These components are assembled into expression vectors. Expressionvectors for bacteria are well known in the art, and include vectors forBacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcuslividans, among others.

The bacterial expression vectors are transformed into bacterial hostcells using techniques well known in the art, such as calcium chloridetreatment, electroporation, and others.

In one embodiment, MINK3 proteins are produced in insect cells.Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart.

In a preferred embodiment, MINK3 protein is produced in yeast cells.Yeast expression systems are well known in the art, and includeexpression vectors for Saccharomyces cerevisiae, Candida albicans and C.maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis,Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, andYarrowia lipolytica. Preferred promoter sequences for expression inyeast include the inducible GAL1,10 promoter, the promoters from alcoholdehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and theacid phosphatase gene. Yeast selectable markers include ADE2, HIS4,LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; theneomycin phosphotransferase gene, which confers resistance to G418; andthe CUP1 gene, which allows yeast to grow in the presence of copperions.

The MINK3 protein may also be made as a fusion protein, using techniqueswell known in the art. Thus, for example, for the creation of monoclonalantibodies, if the desired epitope is small, the MINK3 protein may befused to a carrier protein to form an immunogen. Alternatively, theMINK3 protein may be made as a fusion protein to increase expression, orfor other reasons. For example, when the MINK3 protein is a MINK3peptide, the nucleic acid encoding the peptide may be linked to othernucleic acid for expression purposes. Similarly, MINK3 proteins of theinvention can be linked to protein labels, such as green fluorescentprotein (GFP), red fluorescent protein (RFP), blue fluorescent protein(BFP), yellow fluorescent protein (YFP), etc.

In one embodiment, the MINK3 nucleic acids, proteins and antibodies ofthe invention are labeled. By “labeled” herein is meant that a compoundhas at least one element, isotope or chemical compound attached toenable the detection of the compound. In general, labels fall into threeclasses: a) isotopic labels, which may be radioactive or heavy isotopes;b) immune labels, which may be antibodies or antigens; and c) colored orfluorescent dyes. The labels may be incorporated into the compound atany position.

In a preferred embodiment, the MINK3 protein is purified or isolatedafter expression. MINK3 proteins may be isolated or purified in avariety of ways known to those skilled in the art depending on whatother components are present in the sample. Standard purificationmethods include electrophoretic, molecular, immunological andchromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography, and chromatofocusing.For example, the MINK3 protein may be purified using a standardanti-MINK3 antibody column. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.For general guidance in suitable purification techniques, see Scopes,R., Protein Purification, Springer-Verlag, NY (1982). The degree ofpurification necessary will vary depending on the use of the MINK3protein. In some instances no purification will be necessary.

Once expressed and purified if necessary, the MINK3 proteins and nucleicacids are useful in a number of applications.

The nucleotide sequences (or their complement) encoding MINK3 proteinshave various applications in the art of molecular biology, includinguses as hybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. MINK3 nucleic acids are alsouseful for the preparation of MINK3 proteins by the recombinanttechniques described herein.

Full-length native sequence MINK3 genes, or portions thereof, may beused as hybridization probes for a cDNA library to isolate other genes(for instance, those encoding naturally-occurring variants of MINK3protein or MINK3 protein from other species) which have a desiredsequence identity to the MINK3 protein coding sequence. Optionally, thelength of the probes will be about 20 to about 50 bases. Thehybridization probes may be derived from the nucleotide sequences hereinor from genomic sequences including promoters, enhancer elements andintrons of native sequences as provided herein. By way of example, ascreening method will comprise isolating the coding region of the MINK3protein gene using the known DNA sequence to synthesize a selected probeof about 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the MINK3 protein gene of the present inventioncan be used to screen libraries of human cDNA, genomic DNA or mRNA todetermine which members of such libraries the probe hybridizes.

Nucleotide sequences encoding a MINK3 protein can also be used toconstruct hybridization probes for mapping the gene which encodes thatMINK3 protein and for the genetic analysis of individuals with geneticdisorders. The nucleotide sequences provided herein may be mapped to achromosome and specific regions of a chromosome using known techniques,such as in situ hybridization, linkage analysis against knownchromosomal markers, and hybridization screening with libraries.

The isolation of mRNA comprises isolating total cellular RNA bydisrupting a cell and performing differential centrifugation. Once thetotal RNA is isolated, mRNA is isolated by making use of the adeninenucleotide residues known to those skilled in the art as a poly (A) tailfound on virtually every eukaryotic mRNA molecule at the 3′ end thereof.Oligonucleotides composed of only deoxythymidine [olgo(dT)] are linkedto cellulose and the oligo(dT)-cellulose packed into small columns. Whena preparation of total cellular RNA is passed through such a column, themRNA molecules bind to the oligo(dT) by the poly (A) tails while therest of the RNA flows through the column. The bound mRNAs are theneluted from the column and collected.

Nucleic acids which encode MINK3 protein or its modified forms can alsobe used to generate either transgenic animals or “knock out” animalswhich, in turn, are useful in the development and screening oftherapeutically useful reagents. A transgenic animal (e.g., a mouse orrat) is an animal having cells that contain a transgene, which transgenewas introduced into the animal or an ancestor of the animal at aprenatal, e.g., an embryonic stage. A transgene is a DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops. In one embodiment, cDNA encoding a MINK3 protein can be usedto clone genomic DNA encoding a MINK3 protein in accordance withestablished techniques and the genomic sequences used to generatetransgenic animals that contain cells which express the desired DNA.Methods for generating transgenic animals, particularly animals such asmice or rats, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically,particular cells would be targeted for the MINK3 protein transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding a MINK3 protein introduced intothe germ line of the animal at an embryonic stage can be used to examinethe effect of increased expression of the desired nucleic acid. Suchanimals can be used as tester animals for reagents thought to conferprotection from, for example, pathological conditions associated withits overexpression. In accordance with this facet of the invention, ananimal is treated with the reagent and a reduced incidence of thepathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition.

Alternatively, non-human homologues of the MINK3 protein can be used toconstruct a MINK3 protein “knock out” animal which has a defective oraltered gene encoding a MINK3 protein as a result of homologousrecombination between the endogenous gene encoding a MINK3 protein andaltered genomic DNA encoding a MINK3 protein introduced into anembryonic cell of the animal. For example, cDNA encoding a MINK3 proteincan be used to clone genomic DNA encoding a MINK3 protein in accordancewith established techniques. A portion of the genomic DNA encoding aMINK3 protein can be deleted or replaced with another gene, such as agene encoding a selectable marker which can be used to monitorintegration. Typically, several kilobases of unaltered flanking DNA(both at the 5′ and 3′ ends) are included in the vector [see e.g.,Thomas, et al., Cell, 51:503 (1987) for a description of homologousrecombination vectors]. The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected[see e.g., Li, et al., Cell, 69:915 (1992)]. The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras [see e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the MINK3 protein.

It is understood that the models described herein can be varied. Forexample, “knock-in” models can be formed, or the models can becell-based rather than animal models.

Nucleic acids encoding MINK3 polypeptides, antagonists or agonists mayalso be used in gene therapy. In gene therapy applications, genes areintroduced into cells in order to achieve in vivo synthesis of atherapeutically effective genetic product, for example for replacementof a defective gene. “Gene therapy” includes both conventional genetherapy where a lasting effect is achieved by a single treatment, andthe administration of gene therapeutic agents, which involves the onetime or repeated administration of a therapeutically effective DNA ormRNA. Antisense RNAs and DNAs can be used as therapeutic agents forblocking the expression of certain genes in vivo. It has already beenshown that short antisense oligonucleotides can be imported into cellswhere they act as inhibitors, despite their low intracellularconcentrations caused by their restricted uptake by the cell membrane.(Zamecnik, et al., Proc. Natl. Acad. Sci. USA, 83:4143-4146 [1986]). Theoligonucleotides can be modified to enhance their uptake, e.g. bysubstituting their negatively charged phosphodiester groups by unchargedgroups.

In one aspect, the present invention provides antisense MINK3 nucleicacids. In a preferred embodiment, the antisense MINK3 nucleic acidcomprises a nucleic acid sequence complementary to the nucleic acidsequence set forth by nucleotides 2804-3187 in SEQ ID NO:1. In anotherpreferred embodiment, the present invention provides antisense MINK3nucleic acids consisting essentially of a nucleic acid sequencecomplementary to the nucleic acid sequence set forth by nucleotides2804-3187 in SEQ ID NO:1.

In a preferred embodiment, the MINK3 antisense nucleic acid willhybridize to MINK3 and MINK1 nucleic acids.

In a preferred embodiment, the MINK3 antisense nucleic acid inhibits oneor more MINK3 protein actvities. In another preferred embodiment, theMINK3 antisense nucleic acid inhibits more than one MINK3 proteinactivity. In a further preferred embodiment, the MINK3 antisense nucleicacid inhibits all MINK3 protein activities, In a preferred embodiment,the MINK3 antisense nucleic acid has an activity shared by dominantnegative MINK3 protein.

In a preferred embodiment, such antisense MINK3 nucleic acids arecapable of inhibiting taxol-induced death in mammalian cells. In apreferred embodiment, such antisense MINK3 nucleic acids are capable ofinhibiting taxol-induced cleavage of Rb. In a preferred embodiment, suchantisense MINK3 nucleic acids are capable of promoting survival inmammalian cells following exposure to taxol.

In a preferred embodiment, such antisense MINK3 nucleic acids arecapable of inhibiting the transcription promoting activity of AP-1 inmammalian cells. AP-1 is well known in the art. AP-1 means activationcomplex-1 and refers to a complex of the known transcription factorsc-JUN and c-FOS. In a preferred embodiment, such MINK3 nucleic acids arecapable of inhibiting transcriptional induction by an AP-1 responseelement. AP-1 response elements are well known in the art.

In a preferred embodiment, such antisense MINK3 nucleic acids arecapable of inhibiting growth factor-induced ERK activity in mammaliancells, preferably EGF-induced ERK activity, preferably in cancer cells.In a preferred embodiment, such antisense MINK3 nucleic acids arecapable of inhibiting growth factor-induced proliferation in mammaliancells, preferably EGF-induced proliferation, preferably in cancer cells.In a preferred embodiment, such antisense MINK3 nucleic acids arecapable of inhibiting growth factor-dependent proliferation in mammaliancells, preferably cancer cells.

In a preferred embodiment, such antisense MINK3 nucleic acids arecapable of inhibiting aberrant cell proliferation involving aberrantactivation of the ERK and/or JNK signal transduction pathways.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau, et al., Trends in Biotechnology, 11:205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu, et al.,J. Biol. Chem., 262:4429-4432 (1987); and Wagner, et al., Proc. Natl.Acad. Sci. USA, 87:3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson, et al., Science 256:808-813 (1992).

In a preferred embodiment, the MINK3 proteins, nucleic acids, variants,modified proteins, cells and/or transgenics containing the said nucleicacids or proteins are used in screening assays. Identification of MINK3proteins provided herein permits the design of drug screening assays forcompounds that bind or interfere with the binding to the MINK3 proteinand for compounds which modulate MINK3 protein activity.

The assays described herein preferably utilize the human MINK3 protein,although other mammalian proteins may also be used, including rodents(mice, rats, hamsters, guinea pigs, etc.), farm animals (cows, sheep,pigs, horses, etc.) and primates. These latter embodiments may bepreferred in the development of animal models of human disease. In someembodiments, as outlined herein, variant or derivative MINK3 proteinsmay be used, including deletion MINK3 proteins as outlined above.

In a preferred embodiment, the methods comprise combining a MINK3protein and a candidate bioactive agent, and determining the binding ofthe candidate agent to the MINK3 protein. In other embodiments, furtherdiscussed below, binding interference or bioactivity is determined.

The term “candidate bioactive agent” or “exogeneous compound” as usedherein describes any molecule, e.g., protein, small organic molecule(small molecule chemical compound), carbohydrates (includingpolysaccharides), polynucleotide, lipids, etc. Small molecule chemicalcompositions are preferred. Generally a plurality of assay mixtures arerun in parallel with different agent concentrations to obtain adifferential response to the various concentrations. Typically, one ofthese concentrations serves as a negative control, i.e., at zeroconcentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons,preferably less than about 2000 daltons, preferably less than about 1800daltons, preferably less than about 1700 daltons, preferably less thanabout 1600 daltons, preferably less than about 1500 daltons, preferablyless than about 1400 daltons, preferably less than about 1300 daltons,preferably less than about 1200 daltons, preferably less than about 1100daltons, preferably less than about 1000 daltons. Candidate agentscomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

In a preferred embodiment, a library of different candidate bioactiveagents are used. Preferably, the library should provide a sufficientlystructurally diverse population of randomized agents to effect aprobabilistically sufficient range of diversity to allow binding to aparticular target. Accordingly, an interaction library should be largeenough so that at least one of its members will have a structure thatgives it affinity for the target. Although it is difficult to gauge therequired absolute size of an interaction library, nature provides a hintwith the immune response: a diversity of 10⁷-10⁸ different antibodiesprovides at least one combination with sufficient affinity to interactwith most potential antigens faced by an organism. Published in vitroselection techniques have also shown that a library size of 10⁷ to 10⁸is sufficient to find structures with affinity for the target. A libraryof all combinations of a peptide 7 to 20 amino acids in length, such asgenerally proposed herein, has the potential to code for 20⁷ (10⁹) to20^(°). Thus, with libraries of 10⁷ to 10⁸ different molecules thepresent methods allow a “working” subset of a theoretically completeinteraction library for 7 amino acids, and a subset of shapes for the20²⁰ library. Thus, in a preferred embodiment, at least 10⁶, preferablyat least 10⁷, more preferably at least 10⁸ and most preferably at least10⁹ different sequences are simultaneously analyzed in the subjectmethods. Preferred methods maximize library size and diversity.

In a preferred embodiment, the candidate bioactive agents are proteins.By “protein” herein is meant at least two covalently attached aminoacids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations. Chemical blocking groups orother chemical substituents may also be added.

In a preferred embodiment, the candidate bioactive agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of procaryotic and eukaryotic proteins may be madefor screening in the systems described herein. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

In a preferred embodiment, the candidate bioactive agents are peptidesof from about 5 to about 30 amino acids, with from about 5 to about 20amino acids being preferred, and from about 7 to about 15 beingparticularly preferred. The peptides may be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. By “randomized” or grammatical equivalents herein ismeant that each nucleic acid and peptide consists of essentially randomnucleotides and amino acids, respectively. Since generally these randompeptides (or nucleic acids, discussed below) are chemically synthesized,they may incorporate any nucleotide or amino acid at any position. Thesynthetic process can be designed to generate randomized proteins ornucleic acids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the candidate bioactive agents are nucleicacids. By “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein means at least two nucleotides covalently linked together. Anucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925(1993) and references therein; Letsinger, J. Org. Chem., 35:3800 (1970);Sprinzl, et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et al.,Nucl. Acids Res., 14:3487 (1986); Sawai, et al., Chem. Lett., 805(1984), Letsinger, et al., J. Am. Chem. Soc. 110:4470 (1988); andPauwels, et al., Chemica Scripta, 26:141 (1986)), phosphorothioate (Mag,et al., Nucleic Acids Res., 19:1437 (1991); and U.S. Pat. No.5,644,048), phosphorodithioate (Briu, et al., J. Am. Chem. Soc.,111:2321 (1989)), O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc. 114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl.,31:1008 (1992); Nielsen, Nature 365:566 (1993); Carlsson, et al.,Nature, 380:207 (1996), all of which are incorporated by reference)).Other analog nucleic acids include those with positive backbones(Denpcy, et al., Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionicbackbones (U.S. Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141;and 4,469,863; Kiedrowshi, et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988);Letsinger, et al., Nucleoside & Nucleotide, 13:1597 (1994); Chapters 2and 3, ASC Symposium Series 580, “Carbohydrate Modifications inAntisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, etal., Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins,et al., Chem. Soc. Rev., pp. 169-176 (1995)). Several nucleic acidanalogs are described in Rawls, C & E News, Jun. 2, 1997, page 35. Allof these references are hereby expressly incorporated by reference.These modifications of the ribose-phosphate backbone may be done tofacilitate the addition of additional moieties such as labels, or toincrease the stability and half-life of such molecules in physiologicalenvironments. In addition, mixtures of naturally occurring nucleic acidsand analogs can be made. Alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. The nucleic acids may be single stranded or doublestranded, as specified, or contain portions of both double stranded orsingle stranded sequence. The nucleic acid may be DNA, both genomic andcDNA, RNA or a hybrid, where the nucleic acid contains any combinationof deoxyribo- and ribo-nucleotides, and any combination of bases,including uracil, adenine, thymine, cytosine, guanine, inosine,xathanine hypoxathanine, isocytosine, isoguanine, etc.

As described above generally for proteins, nucleic acid candidatebioactive agents may be naturally occurring nucleic acids, randomnucleic acids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eukaryotic genomes may be used as is outlined above forproteins.

In a preferred embodiment, the candidate bioactive agents are organicchemical moieties, a wide variety of which are available in theliterature.

In a preferred embodiment, the candidate bioactive agents are linked toa fusion partner. By “fusion partner” or “functional group” herein ismeant a sequence that is associated with the candidate bioactive agent,that confers upon all members of the library in that class a commonfunction or ability. Fusion partners can be heterologous (i.e. notnative to the host cell), or synthetic (not native to any cell).Suitable fusion partners include, but are not limited to: a)presentation structures, which provide the candidate bioactive agents ina conformationally restricted or stable form; b) targeting sequences,which allow the localization of the candidate bioactive agent into asubcellular or extracellular compartment; c) rescue sequences whichallow the purification or isolation of either the candidate bioactiveagents or the nucleic acids encoding them; d) stability sequences, whichconfer stability or protection from degradation to the candidatebioactive agent or the nucleic acid encoding it, for example resistanceto proteolytic degradation; e) dimerization sequences, to allow forpeptide dimerization; or f any combination of a), b), c), d), and e), aswell as linker sequences as needed.

In one embodiment, the screening methods described herein make use ofportions of MINK3 proteins, or MINK3 protein fragments. In a preferredembodiment, MINK3 protein fragments having at least one MINK3bioactivity as described herein are used. MINK3 bioactivity includesbinding activity to Nck, modulation of phosphorylation of JNK and/orERK, modulation of JNK and/or ERK phosphorylation, modulation of JNKand/or ERK activation, modulation of JNK and/or ERK signal transduction,inhibition of taxol-induced Rb cleavage and apoptosis, morphologicalchange, modulation of cell proliferation, and disruption of F-actin. Insome embodiments, the assays described herein utilize isolated MINK3proteins. In other embodiments, cells comprising MINK3 proteins areused. In other embodiments, MINK3 proteins that are cell-free but notisolated, as in a cell lysate, are used.

Generally, in a preferred embodiment of the methods herein, for examplefor binding assays, the MINK3 protein or the candidate agent isnon-diffusibly bound to an insoluble support having isolated samplereceiving areas (e.g. a microtiter plate, an array, etc.). The insolublesupports may be made of any composition to which the compositions can bebound, is readily separated from soluble material, and is otherwisecompatible with the overall method of screening. The surface of suchsupports may be solid or porous and of any convenient shape. Examples ofsuitable insoluble supports include microtiter plates, arrays, membranesand beads. These are typically made of glass, plastic (e.g.,polystyrene), polysaccharides, nylon or nitrocellulose, Teflon™, etc.Microtiter plates and arrays are especially convenient because a largenumber of assays can be carried out simultaneously, using small amountsof reagents and samples. In some cases magnetic beads and the like areincluded. The particular manner of binding of the composition is notcrucial so long as it is compatible with the reagents and overallmethods of the invention, maintains the activity of the composition andis nondiffusable. Preferred methods of binding include the use ofantibodies (which do not sterically block either the ligand binding siteor activation sequence when the protein is bound to the support), directbinding to “sticky” or ionic supports, chemical crosslinking, thesynthesis of the protein or agent on the surface, etc. In someembodiments, Nck protein can be used. Following binding of the proteinor agent, excess unbound material is removed by washing. The samplereceiving areas may then be blocked through incubation with bovine serumalbumin (BSA), casein or other innocuous protein or other moiety. Alsoincluded in this invention are screening assays wherein solid supportsare not used; examples of such are described below.

In a preferred embodiment, the MINK3 protein is bound to the support,and a candidate bioactive agent is added to the assay. Alternatively,the candidate agent is bound to the support and the MINK3 protein isadded. Novel binding agents include specific antibodies, non-naturalbinding agents identified in screens of chemical libraries, peptideanalogs, etc. Of particular interest are screening assays for agentsthat have a low toxicity for human cells. A wide variety of assays maybe used for this purpose, including labeled in vitro protein-proteinbinding assays, electrophoretic mobility shift assays, immunoassays forprotein binding, functional assays (phosphorylation assays, etc.) andthe like.

The determination of the binding of the candidate bioactive agent to theMINK3 protein may be done in a number of ways. In a preferredembodiment, the candidate bioactive agent is labeled, and bindingdetermined directly. For example, this may be done by attaching all or aportion of the MINK3 protein to a solid support, adding a labeledcandidate agent (for example a fluorescent label), washing off excessreagent, and determining whether the label is present on the solidsupport. Various blocking and washing steps may be utilized as is knownin the art.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal, e.g.radioisotope, fluorescers, enzyme, antibodies, particles such asmagnetic particles, chemiluminescers, or specific binding molecules,etc. Specific binding molecules include pairs, such as biotin andstreptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

In some embodiments, only one of the components is labeled. For example,the proteins (or proteinaceous candidate agents) may be labeled attyrosine positions using ¹²⁵I, or with fluorophores. Alternatively, morethan one component may be labeled with different labels; using ¹²⁵I forthe proteins, for example, and a fluorophor for the candidate agents.

In a preferred embodiment, the binding of the candidate bioactive agentis determined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to thetarget molecule (i.e. MINK3 protein), such as an antibody, peptide,binding partner, ligand, etc. In a preferred embodiment the competitoris Nck. Under certain circumstances, there may be competitive binding asbetween the bioactive agent and the binding moiety, with the bindingmoiety displacing the bioactive agent. This assay can be used todetermine candidate agents which interfere with binding between MINK3proteins and Nck. “Interference of binding” as used herein means thatnative binding of the MINK3 protein differs in the presence of thecandidate agent. The binding can be eliminated or can be with a reducedaffinity. Therefore, in one embodiment, interference is caused by, forexample, a conformation change, rather than direct competition for thenative binding site.

In one embodiment, the candidate bioactive agent is labeled. Either thecandidate bioactive agent, or the competitor, or both, is added first tothe protein for a time sufficient to allow binding, if present.Incubations may be performed at any temperature which facilitatesoptimal activity, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high through put screening. Typically between 0.1 and 1 hour willbe sufficient. Excess reagent is generally removed or washed away. Thesecond component is then added, and the presence or absence of thelabeled component is followed, to indicate binding.

In a preferred embodiment, the competitor is added first, followed bythe candidate bioactive agent. Displacement of the competitor is anindication that the candidate bioactive agent is binding to the MINK3protein and thus is capable of binding to, and potentially modulating,the activity of the MINK3 protein. In this embodiment, either componentcan be labeled. Thus, for example, if the competitor is labeled, thepresence of label in the wash solution indicates displacement by theagent. Alternatively, if the candidate bioactive agent is labeled, thepresence of the label on the support indicates displacement.

In an alternative embodiment, the candidate bioactive agent is addedfirst, with incubation and washing, followed by the competitor. Theabsence of binding by the competitor may indicate that the bioactiveagent is bound to the MINK3 protein with a higher affinity. Thus, if thecandidate bioactive agent is labeled, the presence of the label on thesupport, coupled with a lack of competitor binding, may indicate thatthe candidate agent is capable of binding to the MINK3 protein.

A preferred embodiment utilizes differential screening to identify drugcandidates that bind to the native MINK3 protein, but cannot bind tomodified MINK3 proteins. The structure of the MINK3 protein may bemodeled, and used in rational drug design to synthesize agents thatinteract with that site. Drug candidates that affect MINK3 bioactivityare also identified by screening drugs for the ability to either enhanceor reduce the activity of the protein.

Positive controls and negative controls may be used in the assays.Preferably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theprotein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

The proteins and nucleic acids provided herein can also be used forscreening purposes wherein the protein-protein interactions of the MINK3proteins can be identified. Genetic systems have been described todetect protein-protein interactions. The first work was done in yeastsystems, namely the “yeast two-hybrid” system. The basic system requiresa protein-protein interaction in order to turn on transcription of areporter gene. Subsequent work was done in mammalian cells. See Fields,et al., Nature, 340:245 (1989); Vasavada, et al., PNAS USA 88:10686(1991); Fearon, et al., PNAS USA 89:7958 (1992); Dang, et al., Mol.Cell. Biol., 11:954 (1991); Chien, et al., PNAS USA, 88:9578 (1991); andU.S. Pat. Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490, and5,637,463. a preferred system is described in U.S. Ser. Nos. 09/050,863,filed Mar. 30, 1998 and 09/359,081 filed Jul. 22, 1999, entitled“Mammalian Protein Interaction Cloning System”. For use in conjunctionwith these systems, a particularly useful shuttle vector is described inU.S. Ser. No. 09/133,944, filed Aug. 14, 1998, entitled “ShuttleVectors”.

In general, two nucleic acids are transformed into a cell, where one isa “bait” such as the gene encoding a MINK3 protein or a portion thereof,and the other encodes a test candidate. Only if the two expressionproducts bind to one another will an indicator, such as a fluorescentprotein, or a gene product required fro survival or growth, beexpressed. Expression of the indicator indicates when a test candidatebinds to the MINK3 protein and can be identified as an MINK3 protein.Using the same system and the identified MINK3 proteins the reverse canbe performed. Namely, the MINK3 proteins provided herein can be used toidentify new baits, or agents which interact with MINK3 proteins.Additionally, the two-hybrid system can be used wherein a test candidateis added in addition to the bait and the MINK3 protein encoding nucleicacids to determine agents which interfere with the bait, such as Nck,and the MINK3 protein.

In one embodiment, a mammalian two-hybrid system is preferred. Mammaliansystems provide post-translational modifications of proteins which maycontribute significantly to their ability to interact. In addition, amammalian two-hybrid system can be used in a wide variety of mammaliancell types to mimic the regulation, induction, processing, etc. ofspecific proteins within a particular cell type. For example, proteinsinvolved in a disease state (i.e., cancer, apoptosis related disorders)could be tested in the relevant disease cells. Similarly, for testing ofrandom proteins, assaying them under the relevant cellular conditionswill give the highest positive results. Furthermore, the mammalian cellscan be tested under a variety of experimental conditions that may affectintracellular protein-protein interactions, such as in the presence ofhormones, drugs, growth factors and cytokines, radiation,chemotherapeutics, cellular and chemical stimuli, etc., that maycontribute to conditions which can effect protein-protein interactions,particularly those involved in cancer.

Assays involving binding such as the two-hybrid system may take intoaccount non-specific binding proteins (NSB).

Screening for agents that modulate the activity of MINK3 protein mayalso be done. In a preferred embodiment, methods for screening for abioactive agent capable of modulating the activity of MINK3 proteincomprise the steps of adding a candidate bioactive agent to a sample ofa MINK3 protein (or cells comprising a MINK3 protein) and determining analteration in the biological activity of the MINK3 protein. “Modulatingthe activity of a MINK3 protein” includes an increase in activity, adecrease in activity, or a change in the type or kind of activitypresent. Thus, in this embodiment, the candidate agent may bind to MINK3protein (although this may not be necessary), and will alter at leastone MINK3 biological or biochemical activity as described herein. Themethods include both in vitro screening methods, as are generallyoutlined above, and in vivo screening of cells for alterations in thepresence, distribution, activity or amount of MINK3 protein.

In a preferred embodiment, the present invention sets forth methods forscreening for modulators of MINK3 activity. By “MINK3 activity” or“MINK3 protein activity” or grammatical equivalents herein is meant atleast one of the MINK3 protein's biological activities, including, butnot limited to, its ability to bind to Nck, modulate the phosphorylationof JNK and/or ERK, modulate JNK and/or ERK activity, modulate signaltransduction via the JNK and/or ERK pathways, modulate F-actinstability, phosphorylate Gelsolin, inhibit taxol-induced RB cleavage andapoptosis, effect changes in morphology or oppose such effectivechanges, modulate cell proliferation, modulate growth factor-inducedproliferation, modulate growth factor-induced ERK activation, modulateAP-1 induced transcription, modulate transcription induced by AP-1response elements. By “modulate” is meant increase, decrease, or alter.In some embodiments, fragments of the MINK3 protein are preferred,particularly fragments having one or more MINK3 protein activities.

In a preferred embodiment, the activity of the MINK3 protein isdecreased; in another preferred embodiment, the activity of the MINK3protein is increased. Thus, bioactive agents that are antagonists arepreferred in some embodiments, and bioactive agents that are agonistsmay be preferred in other embodiments. As used herein, increased oroverexpressed means an increase of at least 10%, more preferably 25-50%,more preferably 50%-75%, and more preferably at least a 100% to 500%increase over the native state. As used herein, decreased orunderexpressed means a decrease of at least 10%, more preferably 25-50%,more preferably 50%-75%, and more preferably at least a 100% to 500%decrease over the native state, i.e., compared to withoutadministeration of the MINK3 proteins, nucleic acids or candidate agentsas described herein.

In a preferred embodiment, the invention provides methods for screeningfor bioactive agents capable of modulating the activity of an MINK3protein. The methods comprise adding a candidate bioactive agent, asdefined above, to a cell comprising a MINK3 protein. Preferred celltypes include almost any cell, including HeLa cells, 293 cells,MDA-MB-231 cells and Phoenix cells. The cells contain a recombinantnucleic acid that encodes an MINK3 protein. In a preferred embodiment, alibrary of candidate agents are tested on a plurality of cellscomprising a nucleic acid encoding a MINK3 protein.

The activity assays, such as having an effect on Nck binding,cytoskeleton organization, JNK and/or ERK phosphorylation, JNK and/orERK activation, JNK and/or ERK signal transduction, F-actin stability,cell proliferation, survival following taxol treatment, Rb cleavagefollowing taxol treatment, and growth factor-induced ERK activation canbe performed to confirm the activity of MINK3 proteins which havealready been identified by their sequence identity/similarity to thesequences set forth in SEQ ID NOs: 1-6 or hybridization to the sequencesset forth in SEQ ID NOs: 1, 3, or 5, as well as to further confirm theactivity of lead compounds identified as modulators of the MINK3proteins provided herein.

The components provided herein for the assays provided herein may alsobe combined to form kits. The kits can be based on the use of theprotein and/or the nucleic acid encoding the MINK3 proteins. In oneembodiment, other components are provided in the kit. Such componentsinclude one or more of packaging, instructions, antibodies, and labels.Additional assays such as those used in diagnostics are furtherdescribed below.

Using the activity and binding assays provided herein, bioactive agents,preferably small molecule chemical compositions as described herein,that may be used as pharmacological compounds are identified. Compoundswith pharmacological activity are able to enhance or interfere with theactivity of the MINK3 protein. The compounds having the desiredpharmacological activity may be administered in a physiologicallyacceptable carrier to a host, as further described below.

The present discovery relating to the role of MINK3 proteins in cellproliferation thus provides methods and compositions for inducing orpreventing cell proliferation in cells. In a preferred embodiment, theMINK3 proteins, and particularly MINK3 protein fragments, are useful inthe study or treatment of conditions which are mediated by the MINK3proteins, i.e. to diagnose, treat or prevent MINK3 associated disorders.“MINK3 associated disorders” or “disease states” include conditionsinvolving both insufficient or excessive cell proliferation, preferablycancer. In another preferred embodiment, candidate bioactive agents,preferably small molecule chemical compositions as described herein, areuseful in the study or treatment of conditions which are mediated by theMINK3 proteins, i.e. to diagnose, treat or prevent MINK3 associateddisorders, including and preferably cancer.

Thus, in one embodiment, methods and compositions for the modulation ofproliferation in cells or organisms are provided. In one embodiment, themethods comprise administering to a cell or individual in need thereof,a MINK3 protein in a therapeutic amount. Alternatively, an anti-MINK3antibody that reduces or eliminates the biological activity of theendogeneous MINK3 protein is administered. In another embodiment, abioactive agent as identified by the methods provided herein isadministered. Alternatively, the methods comprise administering to acell or individual a recombinant nucleic acid encoding an MINK3 protein.As will be appreciated by those in the art, this may be accomplished inany number of ways. In a preferred embodiment, the activity of MINK3 isincreased by increasing the amount of MINK3 in the cell, for example byoverexpressing the endogeneous MINK3 or by administering a gene encodinga MINK3 protein, using known gene-therapy techniques, for example. In apreferred embodiment, the gene therapy techniques include theincorporation of the exogeneous gene using enhanced homologousrecombination (EHR), for example as described in PCT/US93/103868, herebyincorporated by reference in its entirety.

In a preferred embodiment, MINK3 antisense nucleic acids areadministered to a cell or individual. In a preferred embodiment, MINK3antisense nucleic acid decreases the activity of MINK3 by decreasing theamount of MINK3 mRNA and/or protein in the cell or individual. In apreferred embodiment, such a MINK3 antisense nucleic acid comprises thesequence complement of the nucleic acid sequence set forth bynucleotides 2804-3187 in SEQ ID NO: 1. In another preferred embodiment,such a MINK3 antisense nucleic acid consists essentially of the sequencecomplement of the nucleic acid sequence set forth by nucleotides2804-3187 in SEQ ID NO: 1.

It appears that MINK3 is important in cell cycle regulation. Withoutbeing bound by theory, the present invention provides methods andcompositions for the determination of cell cycle disorders. In oneembodiment, the invention provides methods for identifying cellscontaining variant MINK3 genes comprising determining all or part of thesequence of at least one endogenous MINK3 gene in a cell. As will beappreciated by those in the art, this may be done using any number ofsequencing techniques. In a preferred embodiment, the invention providesmethods of identifying the MINK3 genotype of an individual comprisingdetermining all or part of the sequence of at least one MINK3 gene ofthe individual. This is generally done in at least one tissue of theindividual, and may include the evaluation of a number of tissues ordifferent samples of the same tissue. The method may include comparingthe sequence of the sequenced MINK3 gene to a known MINK3 gene, i.e. awild-type gene.

The sequence of all or part of the MINK3 gene can then be compared tothe sequence of a known MINK3 gene to determine if any differencesexist. This can be done using any number of known sequence identityprograms, such as Bestfit, etc. In a preferred embodiment, the presenceof a difference in the sequence between the MINK3 gene of the patientand the known MINK3 gene is indicative of a disease state or apropensity for a disease state.

In one embodiment, methods for determining cell cycle disorders comprisemeasuring the activity of MINK3 in a tissue from the individual orpatient, which may include a measurement of the amount or specificactivity of a MINK3 protein. This activity is compared to the activityof MINK3 from either an unaffected second individual or from anunaffected tissue from the first individual. When these activities aredifferent, the first individual may be at risk for a cell cycle disordersuch as cancer. In this way, for example, monitoring of various diseaseconditions may be done by monitoring the levels of protein or mRNAtherefore, or by monitoring protein activity. Similarly, expressionlevels and activity levels may correlate to the prognosis.

In one aspect, the expression levels of MINK3 genes are determined indifferent patient samples or cells for which either diagnosis orprognosis information is desired. Gene expression monitoring is done ongenes encoding MINK3 proteins. In one aspect, the expression levels ofMINK3 genes are determined for different cellular states, such as normalcells, cells undergoing apoptosis, cells undergoing transformation, andcancer cells. Thus, differential MINK3 gene expression between differentcell states is determined. By comparing MINK3 gene expression levels incells in different states, information including both up- anddown-regulation of MINK3 genes is obtained, which can be used in anumber of ways. For example, the evaluation of a particular treatmentregime may be evaluated: does a chemotherapeutic drug act to improve thelong-term prognosis in a particular patient, whereby prognosis isdetermined based on MINK3 expression. Similarly, diagnosis may be doneor confirmed by comparing patient samples. Furthermore, these geneexpression levels allow screening of drug candidates with an eye tomimicking or altering a particular expression level. This may be done bymaking biochips comprising sets of important MINK3 genes, such as thoseof the present invention, which can then be used in these screens. Thesemethods can also be done on the protein basis; that is, proteinexpression levels of the MINK3 proteins can be evaluated for diagnosticpurposes or to screen candidate agents. In addition, the MINK3 nucleicacid sequences can be administered for gene therapy purposes, includingthe administration of antisense nucleic acids, or the MINK3 proteinsadministered as therapeutic drugs.

“Differential expression,” or grammatical equivalents as used herein,refers to both qualitative as well as quantitative differences in thegenes' temporal and/or cellular expression patterns within and among thecells. Thus, a differentially expressed gene can qualitatively have itsexpression altered, including an activation or inactivation, in, forexample, normal versus apoptotic cell. That is, genes may be turned onor turned off in a particular state, relative to another state. As isapparent to the skilled artisan, any comparison of two or more statescan be made. Such a qualitatively regulated gene will exhibit anexpression pattern within a state or cell type which is detectable bystandard techniques in one such state or cell type, but is notdetectable in both. Alternatively, the determination is quantitative inthat expression is increased or decreased; that is, the expression ofthe gene is either upregulated, resulting in an increased amount oftranscript, or downregulated, resulting in a decreased amount oftranscript. The degree to which expression differs need only be largeenough to quantify via standard characterization techniques as outlinedbelow, such as by use of Affymetrix GeneChip™ expression arrays,Lockhart, Nature Biotechnology, 14:1675-1680 (1996), hereby expresslyincorporated by reference. Other techniques include, but are not limitedto, quantitative reverse transcriptase PCR, Northern analysis and RNaseprotection.

MINK3 sequences bound to biochips include both nucleic acid and aminoacid sequences as defined herein. In a preferred embodiment, nucleicacid probes to MINK3 nucleic acids (both the nucleic acid sequenceshaving the sequences outlined in SEQ ID NOs:1, 3, and 5 and/or thecomplements thereof) are made. The nucleic acid probes attached to thebiochip are designed to be substantially complementary to the MINK3protein nucleic acids, i.e. the target sequence (either the targetsequence of the sample or to other probe sequences, for example insandwich assays), such that hybridization of the target sequence and theprobes of the present invention occurs. As outlined below, thiscomplementarity need not be perfect; there may be any number of basepair mismatches which will interfere with hybridization between thetarget sequence and the single stranded nucleic acids of the presentinvention. However, if the number of mutations is so great that nohybridization can occur under even the least stringent of hybridizationconditions, the sequence is not a complementary target sequence. Thus,by “substantially complementary” herein is meant that the probes aresufficiently complementary to the target sequences to hybridize undernormal reaction conditions, particularly high stringency conditions, asoutlined herein.

A “nucleic acid probe” is generally single stranded but can be partiallysingle and partially double stranded. The strandedness of the probe isdictated by the structure, composition, and properties of the targetsequence. In general, the nucleic acid probes range from about 8 toabout 100 bases long, with from about 10 to about 80 bases beingpreferred, and from about 30 to about 50 bases being particularlypreferred. In some embodiments, much longer nucleic acids can be used,up to hundreds of bases (e.g., whole genes).

As will be appreciated by those in the art, nucleic acids can beattached or immobilized to a solid support in a wide variety of ways. By“immobilized” and grammatical equivalents herein is meant theassociation or binding between the nucleic acid probe and the solidsupport is sufficient to be stable under the conditions of binding,washing, analysis, and removal as outlined below. The binding can becovalent or non-covalent. By “non-covalent binding” and grammaticalequivalents herein is meant one or more of either electrostatic,hydrophilic, and hydrophobic interactions. Included in non-covalentbinding is the covalent attachment of a molecule, such as, streptavidinto the support and the non-covalent binding of the biotinylated probe tothe streptavidin. By “covalent binding” and grammatical equivalentsherein is meant that the two moieties, the solid support and the probe,are attached by at least one bond, including sigma bonds, pi bonds andcoordination bonds. Covalent bonds can be formed directly between theprobe and the solid support or can be formed by a cross linker or byinclusion of a specific reactive group on either the solid support orthe probe or both molecules. Immobilization may also involve acombination of covalent and non-covalent interactions.

In general, the probes are attached to the biochip in a wide variety ofways, as will be appreciated by those in the art. As described herein,the nucleic acids can either be synthesized first, with subsequentattachment to the biochip, or can be directly synthesized on thebiochip.

The biochip comprises a suitable solid substrate. By “substrate” or“solid support” or other grammatical equivalents herein is meant anymaterial that can be modified to contain discrete individual sitesappropriate for the attachment or association of the nucleic acid probesand is amenable to at least one detection method. As will be appreciatedby those in the art, the number of possible substrates are very large,and include, but are not limited to, glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses,plastics, etc. In general, the substrates allow optical detection and donot appreciably show fluorescence.

In a preferred embodiment, the surface of the biochip and the probe maybe derivatized with chemical functional groups for subsequent attachmentof the two. Thus, for example, the biochip is derivatized with achemical functional group including, but not limited to, amino groups,carboxy groups, oxo groups and thiol groups, with amino groups beingparticularly preferred. Using these functional groups, the probes can beattached using functional groups on the probes. For example, nucleicacids containing amino groups can be attached to surfaces comprisingamino groups, for example using linkers as are known in the art; forexample, homo-or hetero-bifunctional linkers as are well known (see 1994Pierce Chemical Company catalog, technical section on cross-linkers,pages 155-200, incorporated herein by reference). In addition, in somecases, additional linkers, such as alkyl groups (including substitutedand heteroalkyl groups) may be used.

In this embodiment, oligonucleotides, corresponding to the nucleic acidprobe, are synthesized as is known in the art, and then attached to thesurface of the solid support. As will be appreciated by those skilled inthe art, either the 5′ or 3′ terminus may be attached to the solidsupport, or attachment may be via an internal nucleoside.

In an additional embodiment, the immobilization to the solid support maybe very strong, yet non-covalent. For example, biotinylatedoligonucleotides can be made, which bind to surfaces covalently coatedwith streptavidin, resulting in attachment.

Alternatively, the oligonucleotides may be synthesized on the surface,as is known in the art. For example, photoactivation techniquesutilizing photopolymerization compounds and techniques are used. In apreferred embodiment, the nucleic acids can be synthesized in situ,using well known photolithographic techniques, such as those describedin WO 95/25116; WO 95/35505; U.S. Pat. Nos. 5,700,637 and 5,445,934; andreferences cited within, all of which are expressly incorporated byreference; these methods of attachment form the basis of the AffimetrixGeneChip™ technology.

The present invention provides novel methods and compositions forscreening for compositions which modulate MINK3 bioactivities includingNck binding activity, as well as the ability to modulate cytoskeletonorganization, JNK and/or ERK phosphorylation, JNK and/or ERK activation,JNK and/or ERK signal transduction, F-actin stability, cellproliferation, survival following taxol treatment, Rb cleavage followingtaxol treatment, and growth factor-induced ERK activation. As above,this can be done by screening for modulators of MINK3 gene expression orfor modulators of MINK3 protein activity. Gene expression and proteinactivity may be evaluated on an individual gene and protein basis, or byevaluating the effect of drug candidates on a gene expression or proteinexpression profile. In a preferred embodiment, the expression profilesare used, preferably in conjunction with high throughput screeningtechniques to allow monitoring for expression profile genes aftertreatment with a candidate agent.

A variety of assays my be used to evaluate the effects of agents onMINK3 gene expression. In a preferred embodiment, assays may be run onan individual gene or protein level. That is, having identified thebioactivities of MINK3 described herein, candidate bioactive agents maybe screened for the ability to modulate MINK3 gene expression and MINK3bioactivities. “Modulation” thus includes both an increase and adecrease in gene expression or activity. The preferred amount ofmodulation will depend on the original change of the gene expression innormal versus tumor tissue, with changes of at least 10%, preferably50%, more preferably 100-300%, and in some embodiments 300-1000% orgreater. Thus, if a gene exhibits a 4 fold increase in tumor compared tonormal tissue, a decrease of about four fold is desired; a 10 folddecrease in tumor compared to normal tissue makes a 10 fold increase inexpression for a candidate agent desirable, etc. Alternatively, wherethe MINK3 sequence has been altered but shows the same expressionprofile or an altered expression profile, the protein will be detectedas outlined herein.

As will be appreciated by those in the art, this may be done byevaluation at either the gene transcript or the protein level; that is,the amount of gene expression may be monitored using nucleic acid probesand the quantification of gene expression levels, or, alternatively, thelevel of the gene product itself can be monitored, for example throughthe use of antibodies to the MINK3 protein and standard immunoassays.Alternatively, binding and bioactivity assays with the protein may bedone as outlined herein.

In a preferred embodiment, gene expression monitoring is done and anumber of genes, i.e. an expression profile, are monitoredsimultaneously, although multiple protein expression monitoring can bedone as well. For example, protein can be monitored through the use ofantibodies to the MINK3 protein and standard immunoassays (ELISAs, etc.)or other techniques, including mass spectroscopy assays, 2D gelelectrophoresis assays, etc.

In this embodiment, the MINK3 nucleic acid probes are attached tobiochips as outlined herein for the detection and quantification ofMINK3 sequences in a particular cell.

In another method detection of the mRNA is performed in situ. In thismethod permeabilized cells or tissue samples are contacted with adetectably labeled nucleic acid probe for sufficient time to allow theprobe to hybridize with the target mRNA. Following washing to remove thenon-specifically bound probe, the label is detected. For example adigoxygenin labeled riboprobe (RNA probe) that is complementary to themRNA encoding an MINK3 protein is detected by binding the digoxygeninwith an anti-digoxygenin secondary antibody and developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.

In another preferred method, expression of MINK3 protein is performedusing in situ imaging techniques employing antibodies to MINK3 proteins.In this method cells are contacted with from one to many antibodies tothe MINK3 protein(s). Following washing to remove non-specific antibodybinding, the presence of the antibody or antibodies is detected. In oneembodiment the antibody is detected by incubating with a secondaryantibody that contains a detectable label. In another method the primaryantibody to the MINK3 protein(s) contains a detectable label. In anotherpreferred embodiment each one of multiple primary antibodies contains adistinct and detectable label. This method finds particular use insimultaneous screening for a plurality of MINK3 proteins. The label maybe detected in a luminometer which has the ability to detect anddistinguish emissions of different wavelengths. In addition, afluorescence activated cell sorter (FACS) can be used in this method. Aswill be appreciated by one of ordinary skill in the art, numerous otherhistological imaging techniques are useful in the invention and theantibodies can be used in ELISA, immunoblotting (Western blotting),immunoprecipitation, BIACORE technology, and the like.

The present invention provides additional methods and compositions forscreening candidate bioactive agents for the ability to modulate MINK3bioactivities as described herein.

For example, candidate bioactive agents may be screened for the abilityto modulate transcriptional activation by the AP-1 protein complex. Inone embodiment, such a method comprises the steps of combining amammalian cell comprising AP-1 protein complex and a reporter gene fusedto a transcriptional regulatory DNA sequence comprising at least oneAP-1 response element, and a recombinant nucleic acid encoding a MINK3protein, and a candidate bioactive agent, and determining the level ofreporter gene expression in the presence and absence of candidate agent.

In a preferred embodiment, such a reporter gene is luciferase. In apreferred embodiment, such a candidate agent is a small moleculechemical compound. In a preferred embodiment, such a mammalian cell is aHeLa cell, a 293 cell, a Phoenix cell, an MDA-MD-231 cell, or an A549cell.

As another example, candidate bioactive agents may be screened for theability to modulate growth factor-induced ERK activation. In oneembodiment, such a method comprises combining a mammalian cellcomprising a recombinant nucleic acid encoding a MINK3 protein, and acandidate bioactive agent, and determining the level of ERK activationin the presence and absence of candidate agent.

ERK activation may be determined in several ways as will be appreciatedby those in the art. In a preferred embodiment, ERK1 isimmunoprecipitated from cell lysate using anti-ERK antibody, and theimmunoprecipitate is used in an in vitro kinase assay with myelin basicprotein (MBP) as a substrate. In vitro kinase assays are known in theart. In such an in vitro kinase assay, isotopically labeled ATP,preferably γP³²-labeled, may be used as a source of phosphate for thekinase assay. Briefly, MBP is a substrate for activated ERK1. Ifactivated ERK1 is present in the immunoprecipitate, it will catalyze thetransfer of a labeled phosphate group to the substrate, MBP. Theproducts of the in vitro kinase assay may be separated by gelelectrophoresis as is known in the art. In this way, the MBP in thekinase assay mixture may be separated from labeled ATP and otherconstituents in the assay mixture. The amount of isotope incorporated byMBP, indicative of the amount of ERK activity in the immunoprecipitate,may then be determined using techniques known in the art.

In this way, the level of ERK activation in the presence and absence ofcandidate agent may be determined and compared to identify a candidateagent capable of either inhibiting ERK activation, or inducing ERKactivation.

In one embodiment, candidate agents are screened for the ability tomodulate ERK activation in response to growth factors. In thisembodiment, ERK activation may be determined as described above. In apreferred embodiment, candidate agents are screened for the ability tomodulate ERK activation in response to epidermal growth factor (EGF). Inone embodiment, such a method comprises exposing mammalian cellscomprising the EGF receptor to EGF and then determining ERK activationin the presence and absence of candidate agent as described above.

In another embodiment, such a method comprises combining a cellcomprising ERK and MINK3 proteins, as well as comprising a reporter geneunder the control of an ERK responsive transcriptional regulatoryelement (such as an ELK response element, as is known in the art), and acandidate agent, and determining the level of reporter gene expressionin the presence and absence of candidate agent.

As will be appreciated by those in the art, the in vitro kinase assaydescribed above may be used similarly to determine the level of JNKactivation. The present invention thus provides methods for screeningcandidate bioactive agents for the ability to modulate JNK activation.In a preferred embodiment, the level of activation of JNK2 is determinedin the presence and absence of candidate agent. In such a method, JNK2is immunoprecipitated with anti-JNK antibody, and aglutathione-S-transferase:c-JUN fusion protein serves as substrate forJNK in an in vitro kinase assay as described above, wherein the c-JUNmoiety is a substrate for JNK.

As another example, candidate bioactive agents may be screened for theability to modulate JNK and/or ERK phosphorylation. In one embodiment,such a method comprises the steps of combining a candidate agent, and acell comprising a recombinant nucleic acid encoding a MINK3 protein, andERK and/or JNK proteins, and determining the level of phosphorylation ofERK and/or JNK in the presence and absence of candidate agent. As willbe appreciated by those in the art, the determination of ERK and JNKphosphorylation can be done in a number of ways. For example,isotopically-labeled ATP may be added to the cells in order to serve asa source of phosphate for the phosphorylation of JNK and ERK. Followingincubation, ERK and/or JNK may be immunoprecipitated using appropriateantibodies as described above, and the immunoprecipitates may beresolved by gel electrophoresis. The amount of radioactive phosphateassociated with JNK or ERK (at the appropriate molecular weight) is thendetermined using techniques known in the art.

Alternatively, such a method may comprise adding a candidate agent to aMINK3 protein which may be isolated or cell-free as in a cell lysate,and then adding an ERK and/or JNK protein, which may be isolated orcell-free, and determining the level of phosphorylation of ERK and/orJNK.

As another example, candidate bioactive agents may be screened for theability to modulate Rb cleavage in response to exposure to taxol. In apreferred embodiment, such a method comprises combining a mammalian cellcomprising a recombinant nucleic acid encoding a MINK3 protein, and Rb,and a candidate agent, and exposing the cell to taxol, and determiningthe level of Rb cleavage in response to taxol in the presence andabsence of candidate agent. The level of Rb cleavage may be determinedby immunoprecipitating Rb or a portion thereof from cell lysate using ananti-Rb antibody. The immunoprecipitate may then be resolved using gelelectrophoresis. A western blot using anti-Rb antibody may then be doneto determine the molecular weight of the Rb species inimmunoprecipitate. The appearance of an Rb immunoreactive band at amolecular weight lower than that of native Rb indicates Rb cleavage hasoccurred. The relative amounts of cleaved to native Rb determines thelevel of Rb cleavage in the sample.

As another example, candidate bioactive agents may be screened for theability to modulate cell survival in response to exposure to taxol. In apreferred embodiment, such a method comprises combining a mammalian cellcomprising a nucleic acid encoding a MINK3 protein, and a candidatebioactive agent, exposing the cell to taxol, and determining the levelof cell survival in the presence and absence of candidate agent. Thelevel of cell survival may be determined in many ways as will beappreciated by those in the art. For example, enzymatic assays formitochondrial function, such as the MTT or XTT assays, may be used todetermine the level of respiration in cells, an indicator of cellsurvival. Additionally, survival may be inferred from the absence ofwell known indicators of apoptosis, including genomic DNA laddering.

As another example, candidate bioactive agents may be screened for theability to modulate proliferation in mammalian cells. In a preferredembodiment, such a method comprises combining a mammalian cellcomprising a recombinant nucleic acid encoding a MINK3 protein, and acandidate agent, and determining the proliferation of the cell in thepresence and absence of candidate agent. As will be appreciated by thosein the art, mammalian cell proliferation may be determined in many ways.For example, cell density in a sample of mammalian cells may bedetermined over time by measuring the optical density of the sample,preferably at 490 nm. The density of the sample is indicative of thenumber of cells in the sample, which is in turn indicative of the levelof proliferation in the cells of the sample. In a preferred embodiment,A549 cells are used.

As another example, candidate bioactive agents may be screened for theability to modulate F-actin stability. In a preferred embodiment, such amethod comprises combining a mammalian cell comprising a recombinantnucleic acid encoding a MINK3 protein, and a candidate agent, anddetermining the stability of F-actin in the presence and absence ofcandidate agent. As will be appreciated by those in the art, thestability of F-actin may be determined in several ways. For example, theamount of actin in a triton X-100 soluble fraction versus the amount ofactin in a triton X-100 insoluble fraction may be determined using byrunning western blots with the fractions and using an anti-actinantibody (Fu et al., JBC 274:30729-30737). After transfection, cells maybe lysed directly on a plate using 250 μl Triton X-100 lysis buffer (1%Triton X-100, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4) with proteaseinhibitors. Cell lysates are centrifuged at 14,000 RPM for 10 min.Supernatant constitutes the Triton X-100 soluble fraction. Pellets arewashed once with 500 μl Triton X-100 lysis buffer and dissolved in 500μl of 1×SDS sample buffer. DNA is sheared by sonication. This representsthe Triton X-100 insoluble fraction. Triton X-100 soluble and insolublefractions derived from the same number of cells are resolved on SDS-PAGEand blotted with an anti-β-actin mAb to determine the content of F- andG-actin.

Alternatively, immunofluorescence using an anti-actin antibody orlabeled phalloidin may be done on whole cells to visualize actinfilaments in the cells.

As another example, candidate bioactive agents may be screened for theability to modulate cell morphology. In a preferred embodiment, such amethod comprises combining a mammalian cell comprising a recombinantnucleic acid encoding a MINK3 protein, and a candidate agent, anddetermining cell morphology in the presence and absence of candidateagent. As will be appreciated by those in the art, cell morphology maybe determined in many ways. Light microscopy and a variety of cellstains known in the art may be used to visualize cells and determinemorphology.

In preferred embodiments, such a method uses Phoenix cells, 293 cells,or MDA-MB-231 cells.

In a preferred embodiment, the ability of a candidate agent to modulatemorphology is dependent on MEK activity. Dependence on MEK activity canbe determined through the use of the known MEK inhibitor PD98059. Cellmorphology can be determined as described above, in the presence andabsence of candidate agent, and further in the presence and absence ofthe MEK inhibitor PD98059.

In one aspect, the invention is directed to methods for screening for abioactive agent capable of modulating JNK phosphorylation and/oractivation. In one aspect, the invention is directed to methods forscreening for a bioactive agent capable of modulating the JNK signaltransduction pathway. In a preferred embodiment, the methods comprisecontacting a candidate bioactive agent to a mammalian cell comprising arecombinant MINK3 nucleic acid encoding a MINK3 protein and a JNKprotein and determining JNK activity in the presence of candidate agent.In a preferred embodiment, JNK activity is determined in the presenceand absence of candidate agent. The recombinant MINK3 nucleic acid isexpressed in said mammalian cell and will activate JNK protein in theabsence of candidate bioactive agent. In a preferred embodiment, theencoded MINK3 protein comprises an amino acid sequence having at leastabout 90% identity to an amino acid sequence selected from the groupconsisting of the amino acid sequences set forth in SEQ ID NOs:2, 4 and6. A decrease in the activity of JNK protein in the presence ofcandidate bioactive agent indicates that the candidate bioactive agentis capable of modulating JNK activity.

In one aspect, the invention is directed to methods for screening for abioactive agent capable of modulating ERK phosphorylation and/oractivation. In one aspect, the invention is directed to methods forscreening for a bioactive agent capable of modulating the ERK signaltransduction pathway. In a preferred embodiment, the methods comprisecontacting a candidate bioactive agent to a mammalian cell comprising arecombinant MINK3 nucleic acid encoding a MINK3 protein and a ERKprotein and determining ERK activity in the presence of candidate agent.In a preferred embodiment, ERK activity is determined in the presenceand absence of candidate agent. The recombinant MINK3 nucleic acid isexpressed in said mammalian cell and will activate ERK protein in theabsence of candidate bioactive agent. In a preferred embodiment, theencoded MINK3 protein comprises an amino acid sequence having at leastabout 90% identity to an amino acid sequence selected from the groupconsisting of the amino acid sequences set forth in SEQ ID NOs:2, 4 and6. A decrease in the activity of ERK protein in the presence ofcandidate bioactive agent indicates that the candidate bioactive agentis capable of modulating ERK activity.

In a preferred embodiment, the methods comprise contacting a mammaliancell with a growth factor which will activate JNK and/or ERK. In apreferred embodiment, the growth factor used in epidermal growth factor(EGF).

In one embodiment, the MINK3 proteins of the present invention may beused to generate polyclonal and monoclonal antibodies to MINK3 proteins,which are useful as described herein. Similarly, the MINK3 proteins canbe coupled, using standard technology, to affinity chromatographycolumns. These columns may then be used to purify MINK3 antibodies. In apreferred embodiment, the antibodies are generated to epitopes unique tothe MINK3 protein; that is, the antibodies show little or nocross-reactivity to other proteins. These antibodies find use in anumber of applications. For example, the MINK3 antibodies may be coupledto standard affinity chromatography columns and used to purify MINK3proteins as further described below. The antibodies may also be used asblocking polypeptides, as outlined above, since they will specificallybind to the MINK3 protein.

The anti-MINK3 protein antibodies may comprise polyclonal antibodies.Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. The immunizing agent may include the MINK3 protein or afusion protein thereof. It may be useful to conjugate the immunizingagent to a protein known to be immunogenic in the mammal beingimmunized. Examples of such immunogenic proteins include but are notlimited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvantswhich may be employed include Freund's complete adjuvant and MPL-TDMadjuvant (monophosphoryl Lipid a, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

The anti-MINK3 protein antibodies may, alternatively, be monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler, et al., Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The immunizing agent will typically include the MINK3 protein or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur, etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, pp. 51-63 (1987)].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against MINK3protein. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunosorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson, etal., Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, Supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.

Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteina-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison, et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

The anti-MINK3 protein antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones, et al., Nature 321:522-525 (1986); Riechmann, etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones, et al., Nature, 321:522-525 (1986); Riechmann, et al., Nature,332:323-327 (1988); Verhoeyen, et al., Science, 239:1534-1536 (1988)],by substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom, et al, J. Mol.Biol., 227:381 (1991); Marks, et al., J. Mol. Biol., 222:581 (1991)].The techniques of Cole, et al. and Boerner, et al. are also availablefor the preparation of human monoclonal antibodies (Cole, et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985);Boerner, et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, humanantibodies can be made by introducing of human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016, and in the following scientific publications: Marks, et al.,Bio/Technology, 10:779-783 (1992); Lonberg, et al., Nature, 368:856-859(1994); Morrison, Nature, 368:812-13 (1994); Fishwild, et al., NatureBiotechnology, 14:845-51 (1996); Neuberger, Nature Biotechnology, 14:826(1996); Lonberg, et al., Intern. Rev. Immunol., 13:65-93 (1995).

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe MINK3 protein, the other one is for any other antigen, andpreferably for a cell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities[Milstein, et al., Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker, et al., EMBO J.,10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh, et al., Methods in Enzymology, 121:210 (1986).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

The anti-MINK3 protein antibodies of the invention have variousutilities. For example, anti-MINK3 protein antibodies may be used indiagnostic assays for an MINK3 protein, e.g., detecting its expressionin specific cells, tissues, or serum. Various diagnostic assaytechniques known in the art may be used, such as competitive bindingassays, direct or indirect sandwich assays and immunoprecipitationassays conducted in either heterogeneous or homogeneous phases [Zola,Monoclonal Antibodies: a Manual of Techniques, CRC Press, Inc. pp.147-158 (1987)]. The antibodies used in the diagnostic assays can belabeled with a detectable moiety. The detectable moiety should becapable of producing, either directly or indirectly, a detectablesignal. For example, the detectable moiety may be a radioisotope, suchas ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent or chemiluminescentcompound, such as fluorescein isothiocyanate, rhodamine, or luciferin,or an enzyme, such as alkaline phosphatase, beta-galactosidase orhorseradish peroxidase. Any method known in the art for conjugating theantibody to the detectable moiety may be employed, including thosemethods described by Hunter, et al., Nature, 144:945 (1962); David, etal., Biochemistry, 13:1014 (1974); Pain, et al., J. Immunol. Meth.,40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).

Anti-MINK3 protein antibodies also are useful for the affinitypurification of MINK3 protein from recombinant cell culture or naturalsources. In this process, the antibodies against MINK3 protein areimmobilized on a suitable support, such a Sephadex resin or filterpaper, using methods well known in the art. The immobilized antibodythen is contacted with a sample containing the MINK3 protein to bepurified, and thereafter the support is washed with a suitable solventthat will remove substantially all the material in the sample except theMINK3 protein, which is bound to the immobilized antibody. Finally, thesupport is washed with another suitable solvent that will release theMINK3 protein from the antibody.

The anti-MINK3 protein antibodies may also be used in treatment. In oneembodiment, the genes encoding the antibodies are provided, such thatthe antibodies bind to and modulate the MINK3 protein within the cell.

In one embodiment, a therapeutically effective dose of an MINK3 protein,agonist or antagonist is administered to a patient. By “therapeuticallyeffective dose” herein is meant a dose that produces the effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art, adjustments for MINK3 proteindegradation, or antagonist or agonist degradation or metabolism, as wellas systemic versus localized delivery, as well as the age, body weight,general health, sex, diet, time of administration, drug interaction andthe severity of the condition may be necessary, and will beascertainable with routine experimentation by those skilled in the art.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and organisms. Thus themethods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

The administration of the MINK3 protein, agonist or antagonist of thepresent invention can be done in a variety of ways, including, but notlimited to, orally, subcutaneously, intravenously, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, or intraocularly. In some instances, for example,in the treatment of wounds and inflammation, the composition may bedirectly applied as a solution or spray. Depending upon the manner ofintroduction, the compounds may be formulated in a variety of ways. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt. %.

The pharmaceutical compositions of the present invention comprise anMINK3 protein, agonist or antagonist (including antibodies and bioactiveagents as described herein) in a form suitable for administration to apatient. Small molecule chemical compositions as described herein areespecially preferred. In a preferred embodiment, the pharmaceuticalcompositions are in a water soluble form, such as being present aspharmaceutically acceptable salts, which is meant to include both acidand base addition salts. “Pharmaceutically acceptable acid additionsalt” refers to those salts that retain the biological effectiveness ofthe free bases and that are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Particularly preferred are theammonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

Combinations of the compositions may be administered. Moreover, thecompositions may be administered in combination with other therapeutics,including growth factors or chemotherapeutics and/or radiation.Targeting agents (i.e. ligands for receptors on cancer cells) may alsobe combined with the compositions provided herein.

Without being bound by theory, the pharmaceutical compositions providedherein find use in the treatment and/or prohylaxis of cancer,particularly as cancer involves dysregulated cell proliferation,aberrant morphology, and aberrant migration as in metastasis. Furthercancer may involve aberrant JNK and/or ERK phosphorylation, JNK and/orERK activation, and JNK and/or ERK signal transduction.

In one embodiment provided herein, the antibodies are used forimmunotherapy, thus, methods of immunotherapy are provided. By“immunotherapy” is meant treatment of MINK3 protein related disorderswith an antibody raised against a MINK3 protein. As used herein,immunotherapy can be passive or active. Passive immunotherapy, asdefined herein, is the passive transfer of antibody to a recipient(patient). Active immunization is the induction of antibody and/orT-cell responses in a recipient (patient). Induction of an immuneresponse can be the consequence of providing the recipient with an MINK3protein antigen to which antibodies are raised. As appreciated by one ofordinary skill in the art, the MINK3 protein antigen may be provided byinjecting an MINK3 protein against which antibodies are desired to beraised into a recipient, or contacting the recipient with an MINK3protein nucleic acid, capable of expressing the MINK3 protein antigen,under conditions for expression of the MINK3 protein antigen.

In a preferred embodiment, a therapeutic compound is conjugated to anantibody, preferably an MINK3 protein antibody. The therapeutic compoundmay be a cytotoxic agent. In this method, targeting the cytotoxic agentto apoptotic cells or tumor tissue or cells, results in a reduction inthe number of afflicted cells, thereby reducing symptoms associated withapoptosis, cancer MINK3 protein related disorders. Cytotoxic agents arenumerous and varied and include, but are not limited to, cytotoxic drugsor toxins or active fragments of such toxins. Suitable toxins and theircorresponding fragments include diptheria A chain, exotoxin A chain,ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin andthe like. Cytotoxic agents also include radiochemicals made byconjugating radioisotopes to antibodies raised against MINK3 proteins,or binding of a radionuclide to a chelating agent that has beencovalently attached to the antibody.

In a preferred embodiment, MINK3 protein genes are administered as DNAvaccines, either single nucleic acids or combinations of MINK3 proteingenes. Naked DNA vaccines are generally known in the art; see Brower,Nature Biotechnology, 16:1304-1305 (1998). Methods for the use ofnucleic acids as DNA vaccines are well known to one of ordinary skill inthe art, and include placing an MINK3 protein gene or portion of anMINK3 protein nucleic acid under the control of a promoter forexpression in a patient. The MINK3 protein gene used for DNA vaccinescan encode full-length MINK3 proteins, but more preferably encodesportions of the MINK3 proteins including peptides derived from the MINK3protein. In a preferred embodiment a patient is immunized with a DNAvaccine comprising a plurality of nucleotide sequences derived from aMINK3 protein gene. Similarly, it is possible to immunize a patient witha plurality of MINK3 protein genes or portions thereof, as definedherein. Without being bound by theory, following expression of thepolypeptide encoded by the DNA vaccine, cytotoxic T-cells, helperT-cells and antibodies are induced which recognize and destroy oreliminate cells expressing MINK3 proteins.

In a preferred embodiment, the DNA vaccines include a gene encoding anadjuvant molecule with the DNA vaccine. Such adjuvant molecules includecytokines that increase the immunogenic response to the MINK3 proteinencoded by the DNA vaccine. Additional or alternative adjuvants areknown to those of ordinary skill in the art and find use in theinvention.

The following example serves to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that this example in no way serve to limit the true scope ofthis invention, but rather are presented for illustrative purposes. Allreferences cited herein are expressly incorporated by reference in theirentirety. Moreover, all sequences displayed, cited by reference oraccession number in the references are incorporated by reference herein.

EXAMPLE 1

A MINK3 antisense nucleic acid complementary to a MINK3 cDNA from aJurkat cDNA library was identified in a functional screen for nucleicacids capable of inhibiting cell death following exposure of HeLa cellsto taxol (data not shown). The antisense nucleic acid comprises anucleic acid sequence complimentary to that set forth by nucleotides2804-3187 in SEQ ID NO:1.

MINK3 antisense nucleic acid was used to clone and identify threeisoforms of MINK3 (MINK3a, MINK3b, MINK3c, set forth in SEQ ID NOs:1, 3,and 5, respectively).

HeLa cells were transfected with expression vectors encoding MINK3a,MINK3b, Bcl2, or MINK3 antisense nucleic acid complementary to thenucleic acid sequence set forth by nucleotides 2804-3187 in SEQ ID NO:1.As an additional control, cells were transfected with empty vector.

Following transfection, cells were exposed to a range of taxolconcentrations and a mitochondrial respiration assay (XTT) was performedon cell lysates to determine viable cells.

Both Bcl2 and MINK3 antisense nucleic acid had an inhibitory effect ontaxol-induced cell death, while MINK3a and MINK3b did not appear toaffect cell death (FIG. 7).

EXAMPLE 2

Hela cells were transfected with expression vectors encoding either Bcl2or GFP, or an expression vector comprising a MINK3 antisense nucleicacid complementary to the nucleic acid sequence set forth by nucleotides2804-3187 in SEQ ID NO:1. As an additional control, Hela cells weretransfected with empty vector alone.

Following transfection, cells were exposed to taxol at a concentrationof 40 nm or 60 nm. Cells were collected following taxol treatment andcell lysate was obtained. Western blots were run on cell lysates usinganti-RB antibody and anti-Bcl2 antibody. As a control, anti-cdk2antibody was used.

Rb immunodetection was done on lysates from cells exposed or not exposedto taxol. In samples exposed to taxol, a faster migratingRb-immunoreactive band (“cleaved Rb”) was detected in addition to thenormal Rb immunoreactive band. Cleaved Rb was not detectable in cellstransfected with Bcl2 and exposed to taxol. The amount of cleaved Rbformed in response to taxol was dramatically reduced in cellstransfected with MINK3 antisense nucleic acid complementary to thenucleic acid sequence set forth by nucleotides 2804-3187 in SEQ IDNO: 1. GFP and empty expression vectors had no effect on the formationof cleaved Rb in response to taxol (FIG. 10).

EXAMPLE 3

293 cells comprising a luciferase reporter gene fused to a regulatorysequence comprising an AP1 element were transfected with MINK3 antisensenucleic acid complementary to the nucleic acid sequence set forth bynucleotides 2804-3187 in SEQ ID NO: 1, or an MEKK1 expression vector, oran empty expression vector.

Following transfection, luciferase activity was determined using aluminometer, in order to determine the level of expression of thereporter gene.

MEKK1 induced reporter gene expression, while MINK3 antisense nucleicacid inhibited the basal level of reporter gene expression (FIG. 3).

EXAMPLE 4

Northern blot analysis showed that MINK3 mRNA is expressed at differentlevels in a number of human tissues, including spleen, thymus, prostate,testis, ovary, small intestine, colon, PBL, leukocytes, heart, brain,placenta, lung, liver, skeletal muscle, kidney and pancreas (FIG. 11).

EXAMPLE 5

Northern blot analysis showed that MINK3 mRNA is expressed at differentlevels in a number of tumor cell lines, including HL-60, HeLa S3, K-562,MOLT-4, Raji, SW480, A549, and G361 (FIG. 12).

EXAMPLE 6

Cells were cotransfected with expression vectors encoding MINK3a,MINK3b, or MEKK1. As a control, empty vector was used in place ofMINK3a, MINK3b, or MEKK1 expression vectors.

Cell lysates were collected, and JNK and ERK were immunoprecipitated.

Kinase assays were done using JNK immunprecipitate, GST-cjUN assubstrate, and isotopically labeled γ-ATP.

The kinase assay mixture was resolved using gel electrophoresis, and thelevel of isotope incorporated into substrate was determined.

Kinase assays were also done using ERK immunprecipitate, MBP assubstrate, and isotopically labeled γ-ATP.

The kinase assay mixture was resolved using gel electrophoresis, and thelevel of isotope incorporated into substrate was determined.

As a control, western blots “WB” were performed on immunoprecipitatesfrom different samples to compare the amount of JNK2 and ERK1 in eachimmunoprecipitate.

MINK3a, MINK3b, and MEKK1 induced JNK2 activation, as shown by theincreased phosphorylation of GST-cJUN in kinase assays.

MINK3a induced ERK1 activation, as shown by the increasedphosphorylation of MBP in kinase assays (FIG. 13).

EXAMPLE 7

MINK3a interacts with Nck in a yeast two hybrid assay. (data not shown).

EXAMPLE 8

293 cells were transfected with expression vectors encoding MINK3a andNck.

Following transfection, cells lysates were collected, andimmunoprecipitations were done.

Nck associates with MINK3a in 293 cells. (data not shown)

EXAMPLE 9

Phoenix cells were transfected with expression vectors encoding GFP andeither TNIK, TNIK kinase dead variant (TNIK kd), MINK3a or MINK3b.

Fluorescence microscopy was done to visualize the morphology oftransfected cells.

MINK3a caused a morphological change in Phoenix cells. (data not shown)

EXAMPLE 10

MDA-MB-231 cells expressing GFP, MINK3a, MINK3b, or MINK3 antisensenucleic acid complementary to the nucleic acid sequence set forth bynucleotides 2804-3187 in SEQ ID NO:1 were generated.

Light microscopy was used to visualize cell morphology of transfectedcells.

MINK3a caused a morphological change in MDA-MB-231 cells.

When MINK3a expressing clone was treated with the MEK inhibitor PD98059,the cells reverted to their normal morphology, i.e. a morphologycharacteristic of cells not transfected with MINK3a expression vector.

Thus, the morphological change induced by MINK3a appears to beMEK-dependent (FIGS. 14 and 15).

EXAMPLE 11

MDA-MB-231 cells were transfected with expression vectors encodingeither GFP, MINK3a, MINK3b, or empty expression vector.

Immunofluorescence was done on cell cultures using afluorescently-labeled F-actin binding toxin. (data not shown)

EXAMPLE 12

A549 cells were transfected with expression vector encoding either GFP,MINK3a, MINK3b, or MINK3 antisense nucleic acid complementary to thenucleic acid sequence set forth by nucleotides 2804-3187 in SEQ ID NO:1.

Following transfection, cells were grown in low serum (0.5% fetal bovineserum) or high serum (10% fetal bovine serum) for four days.

The optical density of the cultures was determined at 490 nm in order todetermine the cell density and compare proliferation between cultures.

MINK3a inhibited proliferation of this tumor cell line in low serum(FIG. 8).

EXAMPLE 13

Cells comprising an EGF receptor and a luciferase reporter gene fused toa regulatory DNA sequence responsive to ERK activation were transfectedwith expression constructs encoding MINK3a, MINK3b, TNIK, or MINKantisense nucleic acid complementary to the nucleic acid sequence setforth by nucleotides 2804-3187 in SEQ ID NO:1. As a control, cells weretransfected with empty vector.

Following transfection, cells were exposed to EGF. Following exposure toEGF, luciferase activity was determined using a luminometer, in order todetermine reporter gene expression.

MINK3 antisense nucleic acid inhibited the basal level of ERK-mediatedtranscription of the reporter gene, as well as EGF-induced ERK-mediatedexpression of the reporter gene (FIG. 9).

Antibodies and cytokines—Antibodies used in this report include: anti-HAmAb (Babco) and pAb (Santa Cruz Biotechnology); anti-FLAG mAb (Sigma)and pAb (Santa Cruz); anti-Myc mAb (Babco); anti-Traf2 pAb (Santa Cruz);anti-NCK mAb (Transduction Labs); anti-β-actin mAb (Sigma). TNFα waspurchased from Calbiochem.

Plasmid construction—Full length human MINK3 was cloned into pCI(Promega) derived expression vector pYCI under the control of the CMVpromoter with an HA epitope tag (AYPYDVPDYA (SEQ ID NO:7)) inserted onthe N-terminus by PCR. A kinase mutant form of MINK3 was constructedusing the QuikChange mutagenesis kit (Stratagene) with OligosAGCTTGCAGCCATCAGGGTTATGGATGTCAC (SEQ ID NO:8) andGTGACATCCATAACCTTGATGGCTGCAAGCT (SEQ ID NO:9) to change the highlyconserved lysine 54 in the kinase domain to arginine. Full length humanNCK was similarly cloned into pYCl with a FLAG epitope tag at theN-terminus. Myc-JNK2 and Myc-ERK1 were constructed in the pCR3.1 vectorwith a Myc epitope tag (ASMEQKLISEEDLN (SEQ ID NO:10)) inserted on theN-terminus of JNK2 and ERK1, respectively. All the truncation mutantswere constructed by PCR.

Cell culture, transfection of Phoenix-A cells andimmunoprecipitation—Phoenix-A cells (derivatives of 293 cells) (Coligan,et al., Current Protocols in Immunology Supplement], 31:10.28.1-10.28.17(1999)) were grown in Dulbecco's modified Eagle's medium MINK3plementedwith 10% fetal bovine serum. Transfection of Phoenix-A cells wasperformed using the standard calcium phosphate method (Coligan, et al.,Current Protocols in Immunology Supplement]31:10.28.1-10.28.17 (1999)).Either 4×10⁵ cells in a 6-well plate or 3×10⁶ cells in a 100 mm tissueculture dish were seeded 16 hours before transfection. 3 μg of DNA wasused in the transfection for each well of a 6-well plate, and 10 μg DNAwas used for each 100 mm dish. Media was changed 8 hours aftertransfection. Cells were lysed in lysis buffer (1% NP-40, 20 mMTris-HCl, pH 8.0, 150 mM NaCl) with protease inhibitors (BoehringerMannheim) and analyzed 24 hours after transfection. Cell lysates werecleared by centrifugation (14,000 RPM×10 min). For immunoprecipitationstudies, cell lysates (2×10⁶ cells/lane) were rotated with 2-3 μg ofdesired antibodies and 20 μl 50% slurry of protein A Sepharose(Pharmacia) for 1.5 hrs. Immune complexes were precipitated and thepellets washed three times with lysis buffer. Washed precipitates weresubjected to SDS-PAGE analysis and Western blotting. Supersignal andSupersignal West Duro substrates (Piers) were used as detection systemsfor the Western blotting.

In vitro kinase assays—For the JNK in vitro kinase assay, Myc-JNK2 wasco-transfected into Phoenix-A cells with MINK3 mutants, Traf2 or MEKK asdescribed above. 24 hours after transfection, cells were lysed withlysis buffer MINK3plemented with 20 mM β-glycerophospate, 1 mM NaF, 1 mMNa₃VO₄ and protease inhibitors. Myc-JNK2 was precipitated from clarifiedcell lysates with an anti-Myc mAb and the pellets were washed threetimes with lysis buffer and two times with kinase buffer (20 mM HEPES,pH 7.4, 10 mM MnCl₂, 10 mM MgCl₂, 20 mM β-glycerophosphate, 1 mM NaF, 1mM Na₃VO₄, 0.5 mM DTT). For the kinase reactions, immunoprecipitateswere incubated with 1 μg glutathion S-transferase (GST) c-Jun (1-79)(Santa Cruz Biotechnology) in 20 μl kinase buffer MINK3plemented with 1μM PKI peptide (Sigma), 10 μM ATP, 5 μCi γ-P³² ATP for 20 minutes at 30°C. Kinase reactions were stopped by addition of 20 μl 2×SDS samplebuffer (Norvex), heated at 95° C. for 5 minutes and then loaded ontoSDS-PAGE. ERK and p38 in vitro kinase assays were conducted in a similarfashion. For ERK kinase assays, an anti-Myc mAb was used toimmunoprecipitate Myc-ERK1 and Myelin Basic Protein (MBP, Sigma) wasused as an exogenous substrate. For p38 kinase assays, an anti-FLAG mAbwas used to immunoprecipitate FLAG-p38 and GST-ATF2 (Santa Cruz) wasused as an exogenous substrate. For in vitro kinase assays on MINK3, 3μg wild type HA-MINK3 or 3 μg kinase mutant form of HA-MINK3 wasexpressed in Phoenix-A cells and immunoprecipitated with an anti-HAantibody. Immune complexes were subjected to kinase assays as describedabove in the absence or presence of 0.5 μg Gelsolin as an exogenoussubstrate.

Fluorescent microscopy—Phoenix-A cells seeded in 6-well plates wereco-transfected with GFP and MINK3 constructs as described above. 24hours after transfection, cells were observed using a Nikon Eclipse TE300 fluorescent microscope. For detection of apoptosis, Hoechst 33258was added to transfected Phoenix-A cells (final concentration 5 μg/ml)and the cells were incubated for 30 min at 37° C. before microscopicobservation.

To determine kinase activity, a putative kinase mutant form of MINK3,designated as MINK3(KM), was constructed with a conserved lysine(Lys-54) residue in the ATP binding pocket mutated to arginine. An HAepitope tag was inserted on the N-terminal portion of MINK3(WT) andMINK3(KM). Both proteins were transiently expressed in Phoenix-A cells,and the expressed proteins were subjected to immunoprecipitation and anin vitro kinase assay. A strong phosphorylated band at 150 kD wasdetected in the MINK3(WT) expressed lane, but not in the MINK3(KM)expressed lane. Immunoblotting with an anti-HA antibody showed equallevels of expression of both MINK3(WT) and MINK3(KM) at 150 kD.Therefore, the phosphorylated band in the in vitro kinase assayrepresented autophosphorylated MINK3, and the MINK3(KM) mutant wasdeficient in protein kinase activity.

Tissue distribution of MINK3—The expression pattern of the MINK3 messagewas examined by human multi-tissue Northern blot. Since MINK3 sharedhigh homology with NIK, a probe corresponding to nucleotides 1264-2427of MINK3 was used to rule out any potential cross-hybridization. Thisregion shared only 40% amino acid identity with NIK. Three major bandsof sizes 6.5 kb, 7.5 kb and 9.5 kb were detected. Alternative splicingin the coding region described above is unlikely to account for the sizedifferences among the three messages, since the largest isoform is only273 bps bigger than the smallest isoform. Alternative splicing in theuntranslated region or alternative usage of polyA sites could bepossible explanations. This phenomenon is not unique to MINK3. NIK andHGK also have multiple message sizes. MINK3 is ubiquitously expressed,with higher levels of message detected in heart, brain and skeletalmuscle. Interestingly, heart and skeletal muscle predominantly expressedthe 6.5 kb form; placenta, kidney and pancreas predominantly expressedthe 7.5 kb form; brain, lung and liver expressed all three forms at asimilar level. It is currently unknown whether these messages havedifferent functional roles.

Interaction of MINK3 with NCK—The interaction of MINK3 with NCK wasinvestigated in a similar fashion. Following transient expression ofHA-MINK3 in Phoenix-A cells, the cell lysates were immunoprecipitatedwith an anti-HA antibody and blotted with an anti-NCK antibody.Endogenous NCK specifically co-immunoprecipitated with HA-MINK3. To mapthe domains on MINK3 required for this interaction, HA-tagged MINK3mutants were co-expressed with FLAG-tagged NCK and the HA-MINK3 mutantswere immunoprecipitated with an anti-HA antibody. The immune complexeswere then blotted with an anti-FLAG antibody. MINK3(WT), MINK3(N2),MINK3(C1) and MINK3(M) were all able to associate with NCK, suggestingthat the intermediate domain is also sufficient for MINK3 to bind NCK.Neither the GCKH domain nor the kinase domain showed any detectablebinding to NCK. Immunoblotting cell lysates with anti-HA and anti-FLAGantibodies showed equivalent levels of expression of the transfectedproteins.

Activation of JNK2 by MINK3—We further examined whether MINK3 was ableto activate the JNK pathway. 1 μg, 2 μg or 3 μg of MINK3 expressionplasmid was co-transfected into Phoenix-A cells with Myc-JNK2. 24 hoursafter transfection, Myc-JNK2 was immunoprecipitated from cell lysatesand its kinase activity measured using GST-cJun(1-79) as a substrate.Co-transfection of MINK3 enhanced JNK2 kinase activity in a dosedependent fashion. When 3 μg of MINK3 was transfected, JNK2 activity wasenhanced 3-4 fold. A similar magnitude of JNK2 activation was observedwhen cells were treated for 15 minutes with 100 ng/ml of TNF. Alsoconsistent with published result (Natoli, et al., Science, 275:200-203(1997)), TRAF2 potently activated JNK2 activity. The expression levelsof Myc-JNK2 were controlled by immunoblotting cell lysates with ananti-Myc antibody.

To determine whether MINK3 can also activate the ERK and p38 pathways,Myc-ERK1 and FLAG-p38 were co-transfected into Phoenix-A cells withdifferent doses of MINK3. The transfected kinases were thenimmunoprecipitated from cell lysates and the kinase activities measuredusing MBP and GST-ATF2 as exogenous substrates. In contrast to JNK2,neither ERK1 nor p38 was activated by MINK3 overexpression, whileco-transfection of MEKK1 potently activated both kinases. In addition,MINK3 did not activate NF-kB (data not shown).

To further investigate the mechanism of this activation, the cohort ofMINK3 mutants were co-transfected into Phoenix-A cells with Myc-JNK2 andthe ability of these mutants to up-regulate JNK2 kinase activity wasexamined by the in vitro kinase assay. MINK3(WT), MINK3(KM), MINK3(C1)and MINK3(C2) were all able to activate Myc-JNK2, while MINK3(N1),MINK3(N2), MINK3(M) were not. This result suggested that the C-terminalGCKH region is both necessary and sufficient for activation of the JNKpathway, while the kinase domain is dispensable.

NIK was cloned by its ability to interact with the adapter protein NCK.It associated with NCK SH3 domains via two PxxPxR sequences in theintermediate domain, PCPPSR (aa 574-579; SEQ ID NO:11) and PRVPVR (aa611-616; SEQ ID NO:12). Both sequences were required for efficientinteraction (Su, et al., EMBO J., 16:1279-1290 (1997)). Similar to NIK,MINK3 also interacted with NCK via the intermediate domain. However,PCPPSR is not conserved in MINK3. Instead, MINK3 contained two otherPxxPxR sequences, PNLPPR (aa 562-567; SEQ ID NO:13) and PPLPTR (aa647-652; SEQ ID NO:14), in addition to the conserved PKVPQR (aa 670-675;SEQ ID NO:15). MINK3 likely interacted with NCK through the cooperativeinteraction with these three PxxPxR sequences. NCK is an adapter proteininvolved in many growth factor receptor mediated signal transductionpathways (McCarthy, Bioessays, 20:913-921 (1998)). It has been proposedthat the NIK-NCK interaction may recruit NIK to receptor or non-receptortyrosine kinases to regulate MEKK1 (Su, et al., EMBO J., 16:1279-1290(1997)). MINK3 may be recruited in a similar fashion

1. A recombinant nucleic acid, comprising a nucleic acid sequence havingat least 98% identity over the full length to a nucleic acid sequenceselected from the group consisting of the nucleic acid sequences setforth in SEQ ID NOs:1, 3, and 5, wherein said recombinant nucleic acidencodes a misshapen/NIKS-related kinase 3 (MINK3) protein.
 2. Arecombinant nucleic acid according to claim 1, wherein said nucleic acidcomprises a nucleic acid sequence selected from the group consisting ofthe nucleic acid sequences set forth in SEQ ID NOs:1, 3, and
 5. 3. Arecombinant nucleic acid, comprising a nucleic acid sequence thatencodes a misshapen/NIKS-related kinase 3 (MINK3) protein comprising anamino acid sequence having at least 98% identity over the full length toan amino acid selected from the group consisting of the amino acidsequences set forth in SEQ ID NOs:2 and
 4. 4. An expression vectorcomprising the nucleic acid of claims 1 or
 3. 5. An isolated or culturedhost cell comprising the vector of claim
 4. 6. A method of making amisshapen/NIKS-related kinase 3 (MINK3) protein comprising the step ofculturing the host cell of claim 5 under conditions suitable forexpression of the MINK3 protein.
 7. The method of claim 6, furthercomprising the step of isolating the MINK3 protein.
 8. The recombinantnucleic acid of claim 1, wherein the nucleic acid sequence has at least99% identity over the full length to a nucleic acid sequence selectedfrom the group consisting of the nucleic acid sequences set forth in SEQID NOs:1, 3, and
 5. 9. The recombinant nucleic acid of claim 3,comprising a nucleic acid sequence that encodes a MINK3 proteincomprising an amino acid sequence having at least 99% identity over thefull length to an amino acid selected from the group consisting of theamino acid sequences set forth in SEQ ID NOs:2 and
 4. 10. A recombinantnucleic acid comprising a nucleic acid sequence that encodes amisshapen/NIKS-related kinase 3 (MINK3) protein comprising an amino acidselected from the group consisting of the amino acid sequences set forthin SEQ ID NOs:2, 4, and
 6. 11. The recombinant nucleic acid of claim 3,wherein the MINK3 protein activates a Jun N-terminal kinase (JNK)protein.
 12. The recombinant nucleic acid of claim 3, wherein the MINK3protein activates an extracellular signal response kinase (ERK) protein.13. The recombinant nucleic acid of claim 3, wherein the MINK3 proteinbinds to a Nck protein.
 14. A recombinant nucleic acid comprising anucleotide sequence that is complementary to the full length of thenucleic acid sequence of claims 1 or
 3. 15. A recombinant nucleic acidcomprising a nucleotide sequence that is complementary to a nucleic acidsequence sharing at least 98% identity over the full length to thenucleic acid sequence set forth by nucleotides 2804-3187 in SEQ ID NO:1.